Antibodies against LLG polypeptides of the triacylglycerol lipase family

ABSTRACT

Lipoprotein Lipase like polypeptides, nucleic acids encoding said polypeptides, antisense sequences, and antibodies to said polypeptides are disclosed. Also disclosed are methods for the preparation of said polypeptides in a recombinant system and for the use of said polypeptides to screen for agonists and or antagonists of said polypeptides. Also disclosed are methods and compositions for the treatment of disorders of lipid metabolism.

This application claims the benefit of two provisional applicationsunder 35 U.S.C. §119(e), 60/032,254 and 60/032,783, both of which werefiled Dec. 6, 1996, the disclosures of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

This invention relates to polypeptides of the triacylglycerol lipasefamily, to nucleic acids encoding said polypeptides, to antisensesequences derived from said nucleic acids, and to antibodies againstsaid polypeptides. This invention also relates to the preparation ofsaid polypeptides using recombinant technology, and to the use of saidpolypeptides to screen for agonists and or antagonists of saidpolypeptides. This invention also relates to methods for the therapeuticuse of such polypeptides, and of the nucleic acid sequences encoding thesame in pharmaceutical, including gene therapeutic, compositions for thetreatment of disorders of lipid and lipoprotein metabolism.

BACKGROUND OF THE INVENTION

A) Lipids

Lipids are water-insoluble organic biomolecules, which are essentialcomponents of diverse biological functions, including the storage,transport, and metabolism of energy, and membrane structure andfluidity. Lipids are derived from two sources in man and other animals:some lipids are ingested as dietary fats and oils and, other lipids arebiosynthesized by the human or animal. In mammals at least 10% of thebody weight is lipid, the bulk of which is in the form oftriacylglycerols.

Triacylglycerols, also known as triglycerides and triacylglycerides, aremade up of three fatty acids esterified to glycerol. Dietarytriacylglycerols are stored in adipose tissues as a source of energy, orhydrolyzed in the digestive tract by triacylglycerol lipases, the mostimportant of which is pancreatic lipase. Triacylglycerols aretransported between tissues in the form of lipoproteins.

Lipoproteins are micelle-like assemblies found in plasma which containvarying proportions of different types of lipids and proteins (calledapoproteins). There are five main classes of plasma lipoproteins, themajor function of which is lipid transport. These classes are, in orderof increasing density, chylomicrons, very low density lipoproteins(VLDL), intermediate-density lipoproteins (IDL), low densitylipoproteins (LDL), and high density lipoproteins (HDL). Although manytypes of lipid are found associated with each lipoprotein class, eachclass transports predominantly one type of lipid: triacylglycerolsdescribed above are transported in chylomicrons, VLDL, and IDL; whilephospholipids and cholesterol esters are transported in HDL and LDLrespectively.

Phospholipids are di-fatty acid esters of glycerol phosphate, alsocontaining a polar group coupled to the phosphate. Phospholipids areimportant structural components of cellular membranes. Phospholipids arehydrolyzed by enzymes called phospholipases. Phosphatidylcholine, anexemplary phospholipid, is a major component of most eukaryotic cellmembranes.

Cholesterol is the metabolic precursor of steroid hormones and bileacids as well as an essential constituent of cell membranes. In man andother animals, cholesterol is ingested in the diet and also synthesizedby the liver and other tissues. Cholesterol is transported betweentissues in the form of cholesteryl esters in LDLs and otherlipoproteins.

Membranes surround every living cell, and serve as a barrier between theintracellular and extracellular compartments. Membranes also enclose theeukaryotic nucleus, make up the endoplasmic reticulum, and servespecialized functions such as in the myelin sheath that surrounds axons.A typical membrane contains about 40% lipid and 60% protein, but thereis considerable variation. The major lipid components are phospholipids,specifically phosphatidylcholine and phosphatidylethanolamine, andcholesterol. The physicochemical properties of membranes, such asfluidity, can be changed by modification of either the fatty acidprofiles of the phospholipids or the cholesterol content. Modulating thecomposition and organization of membrane lipids also modulatesmembrane-dependent cellular functions, such as receptor activity,endocytosis, and cholesterol flux.

B) Enzymes

The triacylglycerol lipases are a family of enzymes which play severalpivotal roles in the metabolism of lipids in the body. Three members ofthe human triacylglycerol lipase family have been described: pancreaticlipase, lipoprotein lipase, and hepatic lipase (Goldberg, I. J., Le,N.-A., Ginsberg, H. N., Krauss, R. M., and Lindgren, F. T. (1988) J.Clin. Invest. 81, 561-568; Goldberg, I. J., Le, N., Paterniti J. R.,Ginsberg, H. N., Lindgren, F. T., and Brown, W. V. (1982) J. Clin.Invest. 70, 1184-1192; Hide, W. A., Chan, L., and Li, W.-H. (1992) J.Lipid. Res. 33, 167-178). Pancreatic lipase is primarily responsible forthe hydrolysis of dietary lipids. Variants of pancreatic lipase havebeen described, but their physiological role has not been determined(Giller, T. Buchwald, P., Blum-Kaolin, D., and Hunziker, W. (1992) J.Biol. Chem. 267, 16509-16516). Lipoprotein lipase is the major enzymeresponsible for the distribution and utilization of triglycerides in thebody. Lipoprotein lipase hydrolyzes triglycerides in both chylomicronsand VLDL. Hepatic lipase hydrolyzes triglycerides in IDL and HDL, and isresponsible for lipoprotein remodeling. Hepatic lipase also functions asa phospholipase, and hydrolyzes phospholipids in HDL.

Phospholipases play important roles in the catabolism and remodeling ofthe phospholipid component of lipoproteins and the phospholipids ofmembranes. Phospholipases also play a role in the release of arachidonicacid and the subsequent formation of prostaglandins, leukotrienes, andother lipids which are involved in a variety of inflammatory processes.

The lipase polypeptides encoded by these lipase genes are approximately450 amino acids in length with leader signal peptides to facilitatesecretion. The lipase proteins are comprised of two principal domains(Winkler, K., D'Arcy, A., and Hunziker, W. (1990) Nature 343, 771-774).The amino terminal domain contains the catalytic site while the carboxyldomain is believed to be responsible for substrate binding, cofactorassociation, and interaction with cell receptors (Wong, H., Davis, R.C., Nikazy, J., Seebart, K. E., and Schotz, M. C. (1991) Proc. Nail.Acad. Sci. USA 88, 11290-11294; van Tilbeurgh, H., Roussel, A., Lalouel,J.-M., and Cambillau, C. (1994) J. Biol. Chem. 269, 4626-4633; Wong, H.,Davis, R. C., Thuren, T., Goers, J. W., Nikazy, J., Waite, M., andSchotz, M. C. (1994) J. Biol. Chem. 269, 10319-10323; Chappell, D. A.,Inoue, I., Fry, G. L., Pladet, M. W., Bowen, S. L., Iverius, P.-H.,Lalouel, J.-M., and Strickland, D. K. (1994) J. Biol. Chem. 269,18001-18006). The overall level of amino acid homology between membersof the family is 22-65%, with local regions of high homologycorresponding to structural homologies which are linked to enzymaticfunction.

The naturally occurring lipoprotein lipase protein is glycosylated, andglycosylation is necessary for LPL enzymatic activity (Semenkovich, C.F., Luo, C.-C., Nakanishi, M. K., Chen, S.-H., Smith, L C., and Chan L.(1990) J. Biol. Chem. 265, 5429-5433). There are two sites for N-linkedglycosylation in hepatic and lipoprotein lipase and one in pancreaticlipase. Additionally, four sets of cysteines form disulfide bridgeswhich are essential in maintaining structural integrity for enzymaticactivity (Lo, Smith, L. C., and Chan, L. (1995) Biochem. Biophys. Res.Commun. 206, 266-271; Brady, L., Brzozowski, A. M., Derewenda, Z. S.,Dodson, E., Dodson G., Tolley, S., Turkenburg, J. P., Christiansen, L.,Huge-Jensen B., Norskov, L., Thim, L., and Menge, U. (1990) Nature 343,767-770).

Members of the triacylglycerol lipase family share a number of conservedstructural features. One such feature is the “GXSXG” motif, in which thecentral serine residue Is one of the three residues comprising the“catalytic triad” (Winkler, K., D'Arcy, A., and Hunziker, W. (1990)Nature 343, 771-774; Faustinella, F., Smith, L. C., and Chan, L. (1992)Biochemistry 31, 7219-7223). Conserved aspartate and histidine residuesmake up the balance of the catalytic triad. A short span of 19-23 aminoacids (the “lid region”) forms an amphipathic helix structure and coversthe catalytic pocket of the enzyme (Winkler, K., D'Arcy, A., andHunziker, W. (1990) Nature 343, 771-774). This region diverges betweenmembers of the family, and it has recently been determined that the spanconfers substrate specificity to the enzymes (Dugi, K. A., Dichek H. L.,and Santamarina-Fojo, S. (1995) J. Biol. Chem. 270, 25396-25401).Comparisons between hepatic and lipoprotein lipase have demonstratedthat differences in triacylglycerol lipase and phospholipase activitiesof the enzymes are in part mediated by this lid region (Dugi, K. A.,Dichek H. L., and Santamarina-Fojo, S. (1995) J. Biol. Chem. 270,25396-25401).

The triacylglycerol lipases possess varying degrees of heparin bindingactivity. Lipoprotein lipase has the highest affinity for heparin, andthis binding activity has been mapped to stretches of positively chargedresidues in the amino terminal domain (Ma, Y., Henderson, H. E., Liu,M.-S., Mang, H., Forsythe, I. J., Clarke-Lewis, I., Hayden, M. R., andBrunzell, J. D. J. Lipid Res. 35, 2049-2059). The localization oflipoprotein lipase to the endothelial surface (Cheng, C. F., Oosta, G.M., Bensadoun, A., and Rosenberg, R. D. (1981) J. Biol. Chem. 256,12893-12896) is primarily mediated through binding to surfaceproteoglycans (Shimada K., Gill, P. J., Silbert, J. E., Douglas, W. H.J., and Fanburg, B. L. (1981) J. Clin. Invest. 68, 995-1002; Saxena, U.,Klein, M. G., and Goldberg, I. J. (1991) J. Biol. Chem. 266,17516-17521; Eisenberg, S., Sehayek, E., Olivecrona, T., and Vlodaysky,I. (1992) J. Clin Invest. 90, 2013-2021). It is this binding activitywhich allows the enzyme to accelerate LDL uptake by acting as a bridgebetween LDL and the cell surface (Mulder, M., Lombardi, P., Jansen, H.,vanBerkel T. J., Frants R. R., and Havekes, L. M. (1992) Biochem.Biophys. Res. Comm. 185, 582-587; Rutledge, J. C., and Goldberg, I. J.,(1994) J. Lipid Res. 35. 1152-1160; Tsuchiya, S., Yamabe, M., Yamaguchi,T., Kobayashi, Y., Konno, T., and Tada, K. (1980) Int. J. Cancer 26,171-176).

Lipoprotein lipase and hepatic lipase are both known to function inconjunction with co-activator proteins: apolipoprotein CII forlipoprotein lipase and colipase for pancreatic lipase.

The genetic sequences encoding human pancreatic lipase, hepatic lipaseand lipoprotein lipase have been reported (Genbank accession #M93285,#J03540, and #M15856 respectively). The messenger RNAs of human hepaticlipase and pancreatic lipase are approximately 1.7 and 1.8 kilobases inlength respectively. Two mRNA transcripts of 3.6 and 3.2 kilobases areproduced from the human lipoprotein lipase gene. These two transcriptsutilize alternate polyadenylation signals, and differ in theirtranslational efficiency (Ranganathan, G., Ong, J. M., Yukht, A.,Saghlzadeh, M., Simsolo, R. B., Pauer, A., and Kern, P. A. (1995) J.Biol. Chem. 270, 7149-7155).

C) Physiological Processes

The metabolism of lipids involves the interaction of lipids,apoproteins, lipoproteins, and enzymes.

Hepatic lipase and lipoprotein lipase are multifunctional proteins whichmediate the binding, uptake, catabolism, and remodeling of lipoproteinsand phospholipids. Lipoprotein lipase and hepatic lipase function whilebound to the luminal surface of endothelial cells in peripheral tissuesand the liver respectively. Both enzymes participate in reversecholesterol transport, which is the movement of cholesterol fromperipheral tissues to the liver either for excretion from the body orfor recycling. Genetic defects in both hepatic lipase and lipoproteinlipase are known to be the cause of familial disorders of lipoproteinmetabolism. Defects in the metabolism of lipoproteins result in seriousmetabolic disorders, including hypercholesterolemia, hyperlipidemia, andatherosclerosis.

Atherosclerosis is a complex, polygenic disease which is defined inhistological terms by deposits (lipid or fibrolipid plaques) of lipidsand of other blood derivatives in blood vessel walls, especially thelarge arteries (aorta, coronary arteries, carotid). These plaques, whichare more or less calcified according to the degree of progression of theatherosclerotic process, may be coupled with lesions and are associatedwith the accumulation in the vessels of fatty deposits consistingessentially of cholesterol esters. These plaques are accompanied by athickening of the vessel wall, hypertrophy of the smooth muscle,appearance of foam cells (lipid-laden cells resulting from uncontrolleduptake of cholesterol by recruited macrophages) and accumulation offibrous tissue. The atheromatous plaque protrudes markedly from thewall, endowing it with a stenosing character responsible for vascularocclusions by atheroma, thrombosis or embolism, which occur in thosepatients who are most affected. These lesions can lead to seriouscardiovascular pathologies such as infarction, sudden death, cardiacinsufficiency, and stroke.

The role of triacylglycerol lipases in vascular pathologies such asatherosclerosis has been an area of intense study (reviewed inOlivecrona, G., and Olivecrona, T. (1995) Curr. Opin. Lipid. 6,291-305). Generally, the action of the triacylglycerol lipases isbelieved to be antiatherogenic because these enzymes lower serumtriacylglycerol levels and promote HDL formation. Transgenic animalsexpressing human lipoprotein lipase or hepatic lipase have decreasedlevels of plasma triglycerides and an increased level of high densitylipoprotein (HDL) (Shimada, M., Shimano, H., Gotoda, T., Yamamoto, K.,Kawamura, M., Inaba, T., Yazaki, t., and Yamada, N. (1993) J. Biol.Chem. 268, 17924-17929; Liu, M.-S., Jirik, F. R., LeBoeuf, R. C.,Henderson, H., Castellani, L. W., Lusis, A. J., ma, Y., Forsythe, I. J.,Zhang, H., Kirk, E., Brunzell, J. D., and Hayden, M. R. (1994) J. Biol.Chem. 269, 11417-11424). Humans with genetic defects resulting indecreased levels of lipoprotein lipase activity have been found to havehypertriglyceridernia but no increased risk of coronary heart disease.This is reported to be due to the lack of production ofintermediate-sized, atherogenic lipoproteins which could accumulatewithin the subendothelial space (Zilversmit, D. B. (1973) Circ. Res. 33,633-638).

In the localized area of an atherosclerotic lesion, however, theincreased level of lipase activity is hypothesized to accelerate theatherogenic process (Zilversmit, D. B. (1995) Clin. Chem. 41, 153-158;Zambon, A., Torres, A., Bijvoet, S., Gagne, C., Moojani, S., Lupien, P.J., Hayden M. R., and Brunzell, J. D. (1993) Lancet 341, 1119-1121).This may be due to an increase in the binding and uptake of lipoproteinsby vascular tissue mediated by lipases (Eisenberg, S., Sehayek, E.,Olivecrona, T. Vlodaysky, I. (1992) J. Clin. Invest. 90, 2013-2021;Tabas, I., Li, I., Brocia R. W., Xu, S. W., Swenson T. L. Williams, K.J. (1993) J. Biol. Chem. 268, 20419-20432; Nordestgaard, B. G., andNielsen, A. G. (1994) Curr. Opin. Lipid. 5, 252-257; Williams, K. J.,and Tabas, I. (1995) Art. Thromb. and Vase. Biol. 15, 551-561).Additionally, a high local level of lipase activity may result incytotoxic levels of fatty acids and lysophosphatidylcholine beingproduced in precursors of atherosclerotic lesions.

Despite the understanding that has evolved regarding the role of lipaseactivity in lipid homeostasis, there nevertheless is a need in the artto identify additional genes coding for proteins that regulate lipidmetabolism.

SUMMARY OF THE INVENTION

The present invention relates to the discovery of a lipase like gene(LLG), its expressed polypeptide products, and compositions and methodsfor their use. The LLG polypeptide binds heparin, has homology to humanlipoprotein lipase and hepatic lipase, and comprises a 39 kD catalyticdomain of the triacylglycerol lipase family. In a further embodiment,the polypeptide has phospholipase A activity.

This invention provides an isolated polypeptide comprising the sequenceSEQ ID NO: 10.

This invention further provides an isolated polypeptide comprising thesequence SEQ ID NO: 8 and having an apparent molecular weight of about55 kD or 68 kJ) on a 10% SDS-PAGE gel.

This invention also provides an isolated polypeptide comprising thesequence SEQ ID. NO: 6 and having an apparent molecular weight of about40 kD on a 10% SDS-PAGE gel.

The invention further provides an antigenic fragment of the LLGpolypeptide.

Another aspect of this invention is an isolated nucleic acid encoding apolypeptide having the aforesaid sequence.

Another aspect of this invention is a vector comprising the aforesaidnucleic acid encoding said polypeptide operably linked to a regulatoryregion, such as a promoter.

Another aspect of this invention is a recombinant cell comprising theabove-described vector.

Another aspect of this invention is a method of preparing a polypeptidewhich comprises culturing recombinant cells containing said polypeptideencoding nucleic acid under conditions permitting the expression of saidpolypeptide.

Another aspect of this invention is an antibody which is capable ofspecifically binding to and/or neutralizing the biological activity ofthe polypeptides according to the invention. Indeed, a furthercharacteristic of a polypeptide of the invention is that it specificallybinds an antibody of the invention, i.e., an antibody specific for anLLG polypeptide.

Another aspect of this invention is a composition comprising apolypeptide, nucleic acid, vector, antisense nucleic acid, or antibodyaccording to the invention and a pharmaceutically acceptable carrier.

Another aspect of this invention is a method of screening for agonistsor antagonists of enzymatic activity exhibited by the polypeptides ofthe present invention comprising contacting potential agonists orantagonists with said polypeptides and a substrate thereof and measuringthe ability of the potential agonists or antagonists to enhance orinhibit activity.

Another aspect of this invention is a method for the enzymatichydrolysis of a phosphatidylcholine ester comprising contacting saidphosphatidylcholine ester with a polypeptide according to the invention.

Another aspect of this invention is a method of treatment for improvingthe serum lipid profile of a human or other animal having an undesirablelipid profile comprising administration thereto of an effective amountof a composition according to the invention.

Another aspect of this invention is a method of treating or preventingatherosclerosis in a human or other animal comprising administrationthereto of an effective amount of a composition according to theinvention.

Other aspects and advantages of the present invention are describedfurther in the drawings and in the following detailed description of thepreferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the sequences (SEQ ID Nos: 17-31) of the primers used inthe exemplified PCR amplifications.

FIG. 2 shows the nucleic acid sequence (SEQ ID NO: 1) and the deducedamino acid sequence (SEQ ID NO: 2) of the differential display RT-PCRproduct containing the lipase like gene cDNA. The sequencescorresponding to the two primers used in the amplification areunderlined. The termination codon and polyadenylation signal are boxed.The GAATTC motifs and flanking sequence are from the pCRII vector intowhich the product was cloned.

FIG. 3 shows the nucleic acid sequence (SEQ ID NO: 3) and the deducedamino acid sequence (SEQ ID NO: 4) of the 5′RACE extension of the LLGcDNA. The sequences corresponding to the two primers used in theamplification are underlined. The GAATTC motifs and flanking sequenceare from the pCRII vector into which the product was cloned.

FIG. 4 shows the sequence (SEQ ID NO: 7) of the cDNA containing thecomplete open reading frame of the lipase Like gene, LLGXL. The startcodon (ATG) and termination codon (TGA) are boxed. The Dial site(TTTAAA) and SrfI site (GCCCGGGC) used in the construction of theexpression vectors are underlined.

FIG. 5 shows the deduced amino acid sequence (SEQ ID NO: 8) of the LLGXLprotein. The predicted signal sequence is underlined.

FIGS. 6A-C show a protein sequence alignment of the members of thetriacylglycerol lipase gene family (SEQ ID Nos: 13-15). Shaded residuesare identical to the LLGXL protein (SEQ ID NO: 8). Gaps were introducedinto the sequences to maximize the alignment values using the CLUSTALprogram.

FIG. 7 shows a northern analysis of LLG mRNA in THP-1 cells. Cells werestimulated with either PMA or PMA and oxidized LDL (PMA+oxLDL). Numbersat the left indicate the positions of RNA standards (in kilobases).

FIG. 8 shows a northern analysis of mRNAs from multiple human tissuesprobed with LLG, lipoprotein lipase (LPL) and human beta actin cDNAs.The position of a 4.4 kilobase RNA standard is indicated to the left ofthe LLG and LPL panels.

FIG. 9 shows a northern analysis of LLG and LPL expression in culturedhuman endothelial cells and THP-1 cells. The cells were eitherunstimulated (not exposed to PMA) or stimulated with PMA.

FIG. 10 shows the sequence of the immunizing peptide (SEQ ID NO: 16) andits relation to the LLGXL protein sequence. The peptide is shown in theshaded box. The terminal cysteine was introduced to aid coupling of thepeptide to the carrier protein.

FIG. 11 shows a western analysis of heparin-Sepharose concentratedproteins from conditioned media from cultured endothelial cells. Theblot was probed with anti-LLG antiserum. The numbers to the leftindicate the positions of protein standards in kilodaltons.

FIG. 12 shows a western analysis of heparin-Sepharose bound proteins inconditioned medium from COS-7 cells transiently transfected with anexpression vector containing a cDNA for LLGN or LLGXL or no DNA (Mock).Proteins from PMA-stimulated endothelial cells (HCAEC+PMA) were includedfor size reference. Numbers to the left indicate the apparent molecularweight of the major immunoreactive proteins as determined by acomparison to protein standards.

FIG. 13 shows the sequence of the rabbit LLG PCR product (RLLG. SEQ, SEQID NO: 12) and the sequence alignment between the rabbit LLG PCR productand the corresponding sequence in the human cDNA (LLG7742A). Identicalnucleotides are shaded.

FIG. 14 shows the phospholipase A activity of human LPL, LLGN, andLLGXL, using a phosphatidylcholine substrate.

FIG. 15 shows the triacylglyceride lipase activity of human LPL, LLGN,and LLGXL, using a triolein substrate.

FIG. 16 shows the hybridization of LLG and LPL probes to genomic DNAsfrom different species.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery of a lipase like gene(LLG) and its expressed polypeptide products. The polypeptide products,members of the triacylglycerol lipase family, comprise an approximately39 kD catalytic domain of the triacylglycerol lipase family, e.g.,having the sequence SEQ ID NO: 10. One embodiment of the presentinvention is the LLGN polypeptide, which has 354 amino acids. A secondembodiment of the present invention is the LLGXL polypeptide, which has500 amino acids and exhibits 43% similarity to human lipoprotein lipaseand 37% similarity to human hepatic lipase. The LLGXL polypeptide hasphospholipase A activity.

The inventors isolated a partial cDNA from mRNA of THP-1 cells which hadbeen exposed to phorbol ester and oxidized LDLs. Following a 5′RACEextension of this partial cDNA, the smaller alternately spliced cDNA wasisolated. A second, larger cDNA was isolated from a human placental cDNAlibrary.

Northern analysis demonstrated that the LLG gene is expressed inendothelial cells. Antisera raised against a peptide predicted from theopen reading frame of the cDNA detected proteins of the predicted sizesfor LLGN and LLGXL in conditioned medium from cultured endothelialcells. Treatment of endothelial cells with phorbol esters resulted inincreased production of LLG at both the mRNA and protein levels. This isthe first member of the triacylglycerol lipase family found to beexpressed by endothelial cells.

A) DEFINITIONS

The following defined terms are used throughout the presentspecification and should be helpful in understanding the scope andpractice of the present invention.

A “polypeptide” is a polymeric compound comprised of covalently linkedamino acid residues. Amino acids have the following general structure:

Amino acids are classified into seven groups on the basis of the sidechain R: (1) aliphatic side chains, (2) side chains containing ahydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) sidechains containing an acidic or amide group, (5) side chains containing abasic group, (6) side chains containing an aromatic ring, and (7)proline, an imino acid in which the side chain is fused to the aminogroup.

A “protein” is a polypeptide which plays a structural or functional rolein a living cell.

The polypeptides and proteins of the invention may be glycosylated orunglycosylated.

“Homology” means similarity of sequence reflecting a common evolutionaryorigin. Polypeptides or proteins are said to have homology, orsimilarity, if a substantial number of their amino acids are either (1)identical, or (2) have a chemically similar R side chain. Nucleic acidsare said to have homology if a substantial number of their nucleotidesare identical.

“Isolated polypeptide” or “isolated protein” is a polypeptide or proteinwhich is substantially free of those compounds that are normallyassociated therewith in its natural state (e.g., other proteins orpolypeptides, nucleic acids, carbohydrates, lipids). “Isolated” is notmeant to exclude artificial or synthetic mixtures with other compounds,or the presence of impurities which do not interfere with biologicalactivity, and which may be present, for example, due to incompletepurification, addition of stabilizers, or compounding into apharmaceutically acceptable preparation.

A molecule is “antigenic” when it is capable of specifically interactingwith an antigen recognition molecule of the immune system, such as animmunoglobulin (antibody) or T cell antigen receptor. An antigenicpolypeptide contains at least about 5, and preferably at least about 10,amino acids. An antigenic portion of a molecule can be that portion thatis immunodominant for antibody or T cell receptor recognition, or it canbe a portion used to generate an antibody to the molecule by conjugatingthe antigenic portion to a carrier molecule for immunization. A moleculethat is antigenic need not be itself immunogenic, i.e., capable ofeliciting an immune response without a carrier.

“LLGN polypeptide” and “LLGN protein” mean a polypeptide including thesequence SEQ ID NO: 6, said polypeptide being glycosylated ornon-glycosylated.

“LLGXL polypeptide” and “LLGXL protein” mean a polypeptide including thesequence SEQ ID NO: 8, said polypeptide being glycosylated ornon-glycosylated.

“LLG polypeptide” generically describes both the LLGN polypeptide andthe LLGXL polypeptide.

The LLG polypeptide or protein of the invention includes any analogue,fragment, derivative, or mutant which is derived from an LLG polypeptideand which retains at least one biological property of the LLGpolypeptide. Different variants of the LLG polypeptide exist in nature.These variants may be allelic variations characterized by differences inthe nucleotide sequences of the structural gene coding for the protein,or may involve differential splicing or post-translational modification.The skilled artisan can produce variants having single or multiple aminoacid substitutions, deletions, additions, or replacements. Thesevariants may include, inter alia: (a) variants in which one or moreamino acid residues are substituted with conservative ornon-conservative amino acids, (b) variants in which one or more aminoacids are added to the LLG polypeptide, (c) variants in which one ormore of the amino acids includes a substituent group, and (d) variantsin which the LLG polypeptide is fused with another polypeptide such asserum albumin. Other LLG polypeptides of the invention include variantsin which amino acid residues from one species are substituted for thecorresponding residue in another species, either at conserved ornon-conserved positions. In another embodiment, amino acid residues atnon-conserved positions are substituted with conservative ornon-conservative residues. The techniques for obtaining these variants,including genetic (suppressions, deletions, mutations, etc.), chemical,and enzymatic techniques, are known to persons having ordinary skill inthe art.

If such allelic variations, analogues, fragments, derivatives, mutants,and modifications, including alternative mRNA splicing forms andalternative post-translational modification forms result in derivativesof the LLG polypeptide which retain any of the biological properties ofthe LLG polypeptide, they are included within the scope of thisinvention.

A “nucleic acid” is a polymeric compound comprised of covalently linkedsubunits called nucleotides. Nucleic acid includes polyribonucleic acid(RNA) and polydeoxyribonucleic acid (DNA), both of which may besingle-stranded or double-stranded. DNA includes cDNA, genomic DNA,synthetic DNA, and semi-synthetic DNA. The sequence of nucleotides thatencodes a protein is called the sense sequence.

An “antisense nucleic acid” is a sequence of nucleotides that iscomplementary to the sense sequence. Antisense nucleic acids can be usedto down regulate or block the expression of the polypeptide encoded bythe sense strand.

“Isolated nucleic acid” means a nucleic acid which is substantially freeof those compounds that are normally associated therewith in its naturalstate. “Isolated” is not meant to exclude artificial or syntheticmixtures with other compounds, or the presence of impurities which donot interfere with biological activity, and which may be present, forexample, due to incomplete purification, addition of stabilizers, orcompounding into a pharmaceutically acceptable preparation.

The phrase “a nucleic acid which hybridizes at high stringency” meansthat the hybridized nucleic acids are able to withstand a washing underhigh stringency conditions. An example of high stringency washingconditions for DNA-DNA hybrids is 0.1×SSC, 0.5% SDS at 68° C. Otherconditions of high stringency washing are known to persons havingordinary skill in the art.

“Regulatory region” means a nucleic acid sequence which regulates theexpression of a nucleic acid. A regulatory region may include sequenceswhich are naturally responsible for expressing a particular nucleic acid(a homologous region) or may include sequences of a different origin(responsible for expressing different proteins or even syntheticproteins). In particular, the sequences can be sequences of eukaryoticor viral genes or derived sequences which stimulate or represstranscription of a gene in a specific or non-specific manner and in aninducible or non-inducible manner. Regulatory regions include origins ofreplication, RNA splice sites, enhancers, transcriptional terminationsequences, signal sequences which direct the polypeptide into thesecretory pathways of the target cell, and promoters.

A regulatory region from a “heterologous source” is a regulatory regionwhich is not naturally associated with the expressed nucleic acid.Included among the heterologous regulatory regions are regulatoryregions from a different species, regulatory regions from a differentgene, hybrid regulatory sequences, and regulatory sequences which do notoccur in nature, but which are designed by one having ordinary skill inthe art.

A “vector” is any means for the transfer of a nucleic acid according tothe invention into a host cell. The term “vector” includes both viraland nonviral means for introducing the nucleic acid into a prokaryoticor eukaryotic cell in vitro, ex vivo or in vivo. Non-viral vectorsinclude plasmids, liposomes, electrically charged lipids (cytofectins),DNA-protein complexes, and biopolymers. Viral vectors includeretrovirus, adeno-associated virus, pox, baculovirus, vaccinia, herpessimplex, Epstein-Barr and adenovirus vectors. In addition to nucleicacid according to the invention, a vector may also contain one or moreregulatory regions, and/or selectable markers useful in selecting,measuring, and monitoring nucleic acid transfer results (transfer towhich tissues, duration of expression, etc.).

A “recombinant cell” is a cell which contains a nucleic acid which isnot naturally present in the cell. “Recombinant cell” includes highereukaryotic cells such as mammalian cells, lower eukaryotic cells such asyeast cells, prokaryotic cells, and archaebacterial cells.

“Pharmaceutically acceptable carrier” includes diluents and fillerswhich are pharmaceutically acceptable for method of administration, aresterile, and may be aqueous or oleaginous suspensions formulated usingsuitable dispersing or wetting agents and suspending agents. Theparticular pharmaceutically acceptable carrier and the ratio of activecompound to carrier are determined by the solubility and chemicalproperties of the composition, the particular mode of administration,and standard pharmaceutical practice.

A “lipase” is a protein which can enzymatically cleave a lipidsubstrate.

A “phospholipase” is a protein which can enzymatically cleave aphospholipid substrate.

A “triacylglycerol lipase” is a protein which can enzymatically cleave atriacylglyceride substrate.

“Phosphatidylcholine” is a glycerol phospholipid having the followingstructure:

R and R′ are the hydrocarbon side chains of fatty acids.Phosphatidylcholine is also known as lecithin.

“Lipid profile” means the set of concentrations of cholesterol,triglyceride, lipoprotein cholesterol and other lipids in the body of ahuman or other animal.

An “undesirable lipid profile” is the condition in which theconcentrations of cholesterol, triglyceride, or lipoprotein cholesterolare outside of the age- and gender-adjusted reference ranges. Generally,a concentration of total cholesterol >200 mg/dl, of plasmatriglycerides >200 mg/dl, of LDL cholesterol >130 mg/dl, of HDLcholesterol <39 mg/dl, or a ratio of total cholesterol to HDLcholesterol >4.0 is considered to be an undesirable lipid profile. Anundesirable lipid profile is associated with a variety of pathologicalconditions, including hyperlipidaemias, diabetes hypercholesterolaemia,atherosclerosis, and other forms of coronary artery disease.

B) POLYPEPTIDES

The present invention provides polypeptides which are members of thetriacylglycerol lipase family, and which comprise a 39 kD catalyticdomain of the triacylglycerol lipase family, e.g., having the sequenceSEQ ID NO: 10. One embodiment of the present invention is an isolatedLLG polypeptide comprising the sequence SEQ ID NO: 6 and having anapparent molecular weight of about 40 kD on a 10% SDS-PAGE gel. Anotherembodiment of the present invention is an isolated LLG polypeptidecomprising the sequence SEQ ID NO: 8 and having an apparent molecularweight of about 55 kD or 68 kD on a 10% SDS-PAGE gel.

The polypeptides and proteins of the present invention may berecombinant polypeptides, natural polypeptides, or syntheticpolypeptides, and may be of human, rabbit, or other animal origin. Thepolypeptides are characterized by a reproducible single molecular weightand/or multiple set of molecular weights, chromatographic response andelution profiles, amino acid composition and sequence, and biologicalactivity.

The polypeptides of the present invention may be isolated from naturalsources, such as placental extracts, human plasma, or conditioned mediafrom cultured cells such as macrophages or endothelial cells, by usingthe purification procedures known to one of skill in the art.

Alternatively, the polypeptides of the present invention may be preparedutilizing recombinant DNA technology, which comprises combining anucleic acid encoding the polypeptide thereof in a suitable vector,inserting the resulting vector into a suitable host cell, recovering thepolypeptide produced by the resulting host cell, and purifying thepolypeptide recovered.

C) NUCLEIC ACIDS

The present invention provides isolated nucleic acids which encode LLGpolypeptides.

The present invention also provides antisense nucleic acids which can beused to down regulate or block the expression of LLG polypeptides invitro, ex vivo or in vivo.

The techniques of recombinant DNA technology are known to those ofordinary skill in the art. General methods for the cloning andexpression of recombinant molecules are described in Maniatis (MolecularCloning, Cold Spring Harbor Laboratories, 1982), and in Ausubel (CurrentProtocols in Molecular Biology, Wiley and Sons, 1987), which areincorporated by reference.

The nucleic acids of the present invention may be linked to one or moreregulatory regions. Selection of the appropriate regulatory region orregions is a routine matter, within the level of ordinary skill in theart. Regulatory regions include promoters, and may include enhancers,suppressors, etc.:

Promoters that may be used in the present invention include bothconstitutive promoters and regulated (inducible) promoters. Thepromoters may be prokaryotic or eukaryotic depending on the host. Amongthe prokaryotic (including bacteriophage) promoters useful for practiceof this invention are lacI, lacZ, T3, T7, lambda P₁, P₁, and trppromoters. Among the eukaryotic (including viral) promoters useful forpractice of this invention are ubiquitous promoters (e.g. HPRT,vimentin, actin, tubulin), intermediate filament promoters (e.g. desmin,neurofilaments, keratin, GFAP), therapeutic gene promoters (e.g. MDRtype, CFTR, factor VIII), tissue-specific promoters (e.g. actin promoterin smooth muscle cells, or Flt and Flk promoters active in endothelialcells), promoters which are preferentially activated in dividing cells,promoters which respond to a stimulus (e.g. steroid hormone receptor,retinoic acid receptor), tetracycline-regulated transcriptionalmodulators, cytomegalovirus immediate-early, retroviral LTR,metallothionein, SV-40, E1a, and MLP promoters. Tetracycline-regulatedtranscriptional modulators and CMV promoters are described in WO96/01313, U.S. Pat. Nos. 5,168,062 and 5,385,839, the contents of whichare incorporated herein by reference.

Preferably, the viral vectors used in gene therapy are replicationdefective, that is, they are unable to replicate autonomously in thetarget cell. In general, the genome of the replication defective viralvectors which are used within the scope of the present invention lack atleast one region which is necessary for the replication of the virus inthe infected cell. These regions can either be eliminated (in whole orin part), be rendered non-functional by any technique known to a personskilled in the art. These techniques include the total removal,substitution (by other sequences, in particular by the inserted nucleicacid), partial deletion or addition of one or more bases to an essential(for replication) region. Such techniques may be performed in vitro (onthe isolated DNA) or in situ, using the techniques of geneticmanipulation or by treatment with mutagenic agents.

Preferably, the replication defective virus retains the sequences of itsgenome which are necessary for encapsidating the viral particles.

The retroviruses are integrating viruses which infect dividing cells.The retrovirus genome includes two LTRs, an encapsidation sequence andthree coding regions (gag, pol and env). The construction of recombinantretroviral vectors has been described: see, in particular, EP 453242,EP178220, Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick,BioTechnology 3 (1985) 689, etc. In recombinant retroviral vectors, thegag, pol and env genes are generally deleted, in whole or in part, andreplaced with a heterologous nucleic acid sequence of interest. Thesevectors can be constructed from different types of retrovirus, such as,MoMuLV (“murine Moloney leukaemia virus” MSV (“murine Moloney sarcomavirus”), HaSV (“Harvey sarcoma virus”); SNV (“spleen necrosis virus”);RSV (“Rous sarcoma virus”) and Friend virus.

In general, in order to construct recombinant retroviruses containing asequence encoding LLG according to the invention, a plasmid isconstructed which contains the LTRs, the encapsidation sequence and thecoding sequence. This construct is used to transfect a packaging cellline, which cell line is able to supply in trans the retroviralfunctions which are deficient in the plasmid. In general, the patkagingcell lines are thus able to express the gag, pol and env genes. Suchpackaging cell lines have been described in the prior art, in particularthe cell line PA317 (U.S. Pat. No. 4,861,719); the PsiCRIP cell line(WO90/02806) and the GP+envAm-12 cell line (WO89/07150). In addition,the recombinant retroviral vectors can contain modifications within theLTRs for suppressing transcriptional activity as well as extensiveencapsidation sequences which may include a part of the gag gene (Benderet al., J. Virol. 61 (1987) 1639). Recombinant retroviral vectors arepurified by standard techniques known to those having ordinary skill inthe art.

The adeno-associated viruses (AAV) are DNA viruses of relatively smallsize which can integrate, in a stable and site-specific manner, into thegenome of the cells which they infect. They are able to infect a widespectrum of cells without inducing any effects on cellular growth,morphology or differentiation, and they do not appear to be involved inhuman pathologies. The AAV genome has been cloned, sequenced andcharacterized. It encompasses approximately 4700 bases and contains aninverted terminal repeat (1TR) region of approximately 145 bases at eachend, which serves as an origin of replication for the virus. Theremainder of the genome is divided into two essential regions whichcarry the encapsidation functions: the left-hand part of the genome,which contains the rep gene involved in viral replication and expressionof the viral genes; and the right-hand part of the genome, whichcontains the cap gene encoding the capsid proteins of the virus.

The use of vectors derived from the AAVs for transferring genes in vitroand in vivo has been described (see WO 91/18088; WO 93/09239; U.S. Pat.No. 4,797,368, U.S. Pat. No. 5,139,941, EP 488 528). These publicationsdescribe various AAV-derived constructs in which the rep and/or capgenes are deleted and replaced by a gene of interest, and the use ofthese constructs for transferring the said gene of interest in vitro(into cultured cells) or in vivo, (directly into an organism). Thereplication defective recombinant AAVs according to the invention can beprepared by cotransfecting a plasmid containing the nucleic acidsequence of interest flanked by two AAV inverted terminal repeat (ITR)regions, and a plasmid carrying the AAV encapsidation genes (rep and capgenes), into a cell line which is infected with a human helper virus(for example an adenovirus). The AAV recombinants which are produced arethen purified by standard techniques. The invention also relates,therefore, to an AAV-derived recombinant virus whose genome encompassesa sequence encoding an LLG polypeptide flanked by the AAV ITRs. Theinvention also relates to a plasmid encompassing a sequence encoding anLLG polypeptide flanked by two ITRs from an AAV. Such a plasmid can beused as it is for transferring the LLG sequence, with the plasmid, whereappropriate, being incorporated into a liposomal vector (pseudo-virus).

In a preferred embodiment, the vector is an adenovirus vector.

Adenoviruses are eukaryotic DNA viruses that can be modified toefficiently deliver a nucleic acid of the invention to a variety of celltypes.

Various serotypes of adenovirus exist. Of these serotypes, preference isgiven, within the scope of the present invention, to using type 2 ortype 5 human adenoviruses (Ad 2 or Ad 5) or adenoviruses of animalorigin (see WO94/26914). Those adenoviruses of animal origin which canbe used within the scope of the present invention include adenovirusesof canine, bovine, murine (example: Mavl, Beard et al., Virology 75(1990) 81), ovine, porcine, avian, and simian (example: SAV) origin.Preferably, the adenovirus of animal origin is a canine adenovirus, morepreferably a CAV2 adenovirus (e.g. Manhattan or A26/61 strain (ATCCVR-800), for example).

Preferably, the replication defective adenoviral vectors of theinvention comprise the ITRs, an encapsidation sequence and the nucleicacid of interest. Still more preferably, at least the E1 region of theadenoviral vector is non-functional. The deletion in the E1 regionpreferably extends from nucleotides 455 to 3329 in the sequence of theAd5 adenovirus. Other regions may also be modified, in particular the E3region (WO95/02697), the E2 region (WO94/28938), the E4 region(WO94/28152, WO94/12649 and WO95/02697), or in any of the late genesL1-L5. Defective retroviral vectors are disclosed in WO95/02697.

In a preferred embodiment, the adenoviral vector has a deletion in theE1 and E4 regions. In another preferred embodiment, the adenoviralvector has a deletion in the E1 region into which the E4 region and thesequence encoding LLG are inserted (see FR94 13355).

The replication defective recombinant adenoviruses according to theinvention can be prepared by any technique known to the person skilledin the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham,EMBO J. 3 (1984) 2917). In particular, they can be prepared byhomologous recombination between an adenovirus and a plasmid whichcarries, inter alfa, the DNA sequence of interest. The homologousrecombination is effected following cotransfection of the saidadenovirus and plasmid into an appropriate cell line. The cell linewhich is employed should preferably (i) be transformable by the saidelements, and (ii) contain the sequences which are able to complementthe part of the genome of the replication defective adenovirus,preferably in integrated form in order to avoid the risks ofrecombination. Examples of cell lines which may be used are the humanembryonic kidney cell line 293 (Graham et al., J. Gen. Virol. 36 (1977)59) which contains the left-band portion of the genome of an Ad5adenovirus (12%) integrated into its genome, and cell lines which areable to complement the E1 and E4 functions, as described in applicationsWO94/26914 and WO95/02697. Recombinant adenoviruses are recovered andpurified using standard molecular biological techniques, which are wellknown to one of ordinary skill in the art.

The down regulation of gene expression using antisense nucleic acids canbe achieved at the translational or transcriptional level. Antisensenucleic acids of the invention are preferably nucleic acid fragmentscapable of specifically hybridizing with all or part of a nucleic acidencoding LLG or the corresponding messenger RNA. These antisense nucleicacids can be synthetic oligonucleotides, optionally modified to improvetheir stability and selectivity. They can also be DNA sequences whoseexpression in the cell produces RNA complementary to all or part of theLLG mRNA. Antisense nucleic acids can be prepared by expression of allor part of a sequence selected from the group consisting of SEQ ID No.2, SEQ ID No. 3, SEQ ID No. 7, or SEQ ID No. 11, in the oppositeorientation, as described in EP 140308. Any length of antisense sequenceis suitable for practice of the invention so long as it is capable ofdown-regulating or blocking expression of LLG. Preferably, the antisensesequence is at least 20 nucleotides in length. The preparation and useof antisense nucleic acids, DNA encoding antisense RNAs and the use ofoligo and genetic antisense is disclosed in WO92/15680, the contents ofwhich are incorporated herein by reference.

D) ANTIBODIES

The present invention provides antibodies against the LLG polypeptide.These antibodies may be monoclonal antibodies or polyclonal antibodies.The present invention includes chimeric, single chain, and humanizedantibodies, as well as Fab fragments and the products of an Fabexpression library.

Polyclonal antibodies may be prepared against an antigenic fragment ofan LLG polypeptide, as described in Example 4A. Antibodies may also begenerated against the intact LLG protein or polypeptide, or against afragment, derivative, or epitope of the protein or polypeptide.Antibodies may be obtained following the administration of the protein,polypeptide, fragment, derivative, or epitope to an animal, using thetechniques and procedures known in the art.

Monoclonal antibodies may be prepared using the method of Mishell, B.B., et al., Selected Methods In Cellular Immunology, (W.H. Freeman, ed.)San Francisco (1980). Briefly, a polypeptide of the present invention isused to immunize spleen cells of Balb/C mice. The immunized spleen cellsare fused with myeloma cells. Fused cells containing spleen and myelomacell characteristics are isolated by growth in HAT medium, a mediumwhich kills both parental cells, but allows the fused products tosurvive and grow.

The monoclonal antibodies of the present invention may be “humanized” toprevent the host from mounting an immune response to the antibodies. A“humanized antibody” is one in which the complementarity determiningregions (CDRs) and/or other portions of the light and/or heavy variabledomain framework are derived from a non-human immunoglobulin, but theremaining portions of the molecule are derived from one or more humanimmunoglobulins. Humanized antibodies also include antibodiescharacterized by a humanized heavy chain associated with a donor oracceptor unmodified light chain or a chimeric light chain, or viceversa. The humanization of antibodies may be accomplished by methodsknown in the art (see, e.g. G. E. Mark and E. A. Padlan, “Chapter 4.Humanization of Monoclonal Antibodies”, The Handbook of ExperimentalPharmacology Vol. 113, Springer-Verlag, New York, 1994). Transgenicanimals may be used to express humanized antibodies.

Techniques known in the art for the production of single chainantibodies can be adapted to produce single chain antibodies to theimmunogenic polypeptides and proteins of the present invention.

The anti-LLG antibodies are useful in assays for detecting orquantitating levels of LLG. In one embodiment, these assays provide aclinical diagnosis and assessment of LLG in various disease states and amethod for monitoring treatment efficacy.

E) METHODS OF SCREENING FOR AGONISTS OR ANTAGONISTS

The present invention provides methods of screening small moleculelibraries or natural product sources for agonists (enhancers orco-activators including proteinaceous co-activators) or antagonists(inhibitors) of LLGXL activity. A potential agonist or antagonist iscontacted with LLGXL protein and a substrate of LLGXL, and the abilityof the potential agonist or antagonist to enhance or inhibit LLGXLactivity is measured.

The LLGXL protein used in the method can be produced recombinantly in avariety of host cells, including mammalian cells (as shown in Example7), baculovirus-infected insect cells, yeast, and bacteria LLGexpression in stably transfected CHO cells can be optimized bymethotrexate amplification of the cells. LLGXL protein can also bepurified from natural sources such as human plasma, placental extracts,or conditioned media from cultured endothelial cells, THP-1 cells, ormacrophages.

The optimization of assay parameters including pH, ion concentrations,temperature, concentration of substrate, and emulsification conditionsare determined empirically by one having ordinary skill in the art.

The fatty acid substituents of the substrates may vary in chain lengthas well as in degree and position of unsaturation. The substrates may beradiolabelled in any of several positions. Phospholipid substrates suchas phosphatidylcholine can be radiolabelled, for example, in the Sn-1 orSn-2 fatty acid position, or in the glycerol, phosphate, or polar headgroup (choline in the case of phosphatidylcholine).

As an alternative to radiolabeled substrates, other classes of labeledsubstrates, such as fluorescent substrates or thio-containingsubstrates, can also be used in the screening methods.

Fluorescent substrates are particularly useful in screening assaysbecause enzymatic catalysis can be measured continuously by measuringfluorescence intensity, without the physical separation (extraction) ofthe products from the substrates. An example of a fluorescentphosphatidylcholine substrate isC₆NBD-PC(1-acyl-2-[6-(nitro-2,1,3-benzoxadiazol-4-yl)amino]caproylphosphatidylcholine.

The thio-containing substrates include1,2-bis(hexanoylthio)-1,2-dideoxy-sn-glycero-3-phosphorylcholine (L. J.Reynolds, W. N. Washburn, R. A. Deems, and E. A. Dennis, 1991. Methodsin Enzymology 197: 3-23; L. Yu and E. A. Dennis, 1991. Methods inEnzymology 197: 65-75; L. A. Wittenauer, K. Shirai, R. L. Jackson, andJ. D. Johnson, 1984. Biochem. Biophys. Res. Commun. 118: 894-901).

F) HYDROLYSIS OF PHOSPHATIDYLCHOLINE ESTERS

The present invention provides a method for the enzymatic hydrolysis ofphosphatidylcholine esters, e.g., for industrial or food processing, orin laundry detergents. The polypeptides of the present invention can beused to hydrolyze phosphatidylcholine esters in solution, or the enzymesmay be bound to a solid support which is then contacted with thesubstrate. This method can be used to produce lysophospholipids and freefatty acids.

G) COMPOSITIONS

The present invention provides compositions in a biologically compatible(biocompatible) solution, comprising the polypeptides, nucleic acids,vectors, and antibodies of the invention. A biologically compatiblesolution is a solution in which the polypeptide, nucleic acid, vector,or antibody of the invention is maintained in an active form, e.g., in aform able to effect a biological activity. For example, a polypeptide ofthe invention would have phospholipase activity; a nucleic acid would beable to replicate, translate a message, or hybridize to a complementarynucleic acid; a vector would be able to transfect a target cell; anantibody would bind a polypeptide of the invention. Generally, such abiologically compatible solution will be an aqueous buffer, e.g., Tris,phosphate, or HEPES buffer, containing salt ions. Usually theconcentration of salt ions will be similar to physiological levels. In aspecific embodiment, the biocompatible solution is a pharmaceuticallyacceptable composition. Biologically compatible solutions may includestabilizing agents and preservatives.

Such compositions can be formulated for administration by topical, oral,parenteral, intranasal, subcutaneous, and intraocular, routes.Parenteral administration is meant to include intravenous injection,intramuscular injection, intraarterial injection or infusion techniques.The composition may be administered parenterally in dosage unitformulations containing standard, well known nontoxic physiologicallyacceptable carriers, adjuvants and vehicles as desired.

The preferred sterile injectable preparations can be a solution orsuspension in a nontoxic parenterally acceptable solvent or diluent.Examples of pharmaceutically acceptable carriers are saline, bufferedsaline, isotonic saline (e.g. monosodium or disodium phosphate, sodium,potassium, calcium or magnesium chloride, or mixtures of such salts),Ringer's solution, dextrose, water, sterile water, glycerol, ethanol,and combinations thereof. 1,3-butanediol and sterile fixed oils areconveniently employed as solvents or suspending media. Any bland fixedoil can be employed including synthetic mono- or di-glycerides. Fattyacids such as oleic acid also find use in the preparation ofinjectables.

The composition medium can also be a hydrogel which is prepared from anybiocompatible or non-cytotoxic (homo or hetero) polymer, such as ahydrophilic polyacrylic acid polymer that can act as a drug absorbingsponge. Such polymers have been described, for example, in applicationWO93/08845, the entire contents of which are hereby incorporated byreference. Certain of them, such as, in particular, those obtained fromethylene and/or propylene oxide are commercially available. A hydrogelcan be deposited directly onto the surface of the tissue to be treated,for example during surgical intervention.

Another preferred embodiment of the present invention relates to apharmaceutical composition comprising a replication defectiverecombinant virus and poloxamer. More specifically, the inventionrelates to a composition comprising a replication defective recombinantvirus comprising a nucleic acid encoding an LLG polypeptide andpoloxamer. A preferred poloxamer is Poloxamer 407, which is commerciallyavailable (BASF, Parsippany, N.J.) and is a non-toxic, biocompaliblepolyol, and is most preferred. A poloxamer impregnated with recombinantviruses may be deposited directly on the surface of the tissue to betreated, for example during a surgical intervention. Poloxamer possessesessentially the same advantages as hydrogel while having a lowerviscosity.

H) METHODS OF TREATMENT

The present invention provides methods of treatment which comprise theadministration to a human or other animal of an effective amount of acomposition of the invention.

Effective amounts may vary, depending on the age, type and severity ofthe condition to be treated, body weight, desired duration of treatment,method of administration, and other parameters. Effective amounts aredetermined by a physician or other qualified medical professional.

Polypeptides according to the invention are generally administered indoses of about 0.01 mg/kg to about 100 mg/kg, preferably about 0.1 mg/kgto about 50 mg/kg, and most preferably about 1 mg/kg to about 10 mg/kgof body weight per day.

Recombinant viruses according to the invention are generally formulatedand administered in the form of doses of between about 10⁴ and about10¹⁴ pfu. In the case of AAVs and adenoviruses, doses of from about 10⁶to about 10¹¹ pfu are preferably used. The term pfu (“plaque-formingunit”) corresponds to the infective power of a suspension of virions andis determined by infecting an appropriate cell culture and measuring thenumber of plaques formed. The techniques for determining the pfu titreof a viral solution are well documented in the prior art.

The present invention provides methods of treating atherosclerosiswherein said atherosclerosis is the result of excess, abnormal orinadequate expression of LLG polypeptide activity.

The present invention further provides methods of treating a human orother animal having an undesirable lipid profile, wherein saidundesirable lipid profile is the result of abnormally high or inadequateexpression of LLG polypeptide activity.

The present invention further provides methods of treating diabetes,hyperlipidemia, intrahepatic cholestasis or other metabolic disorderswherein said diabetes, hyperlipidemia, intrahepatic cholestasis or othermetabolic disorder is the result of abnormally high or inadequateexpression of LLG polypeptide activity.

1) Treatment of Undesirable Lipid Profiles Associated with IncreasedExpression of LLG Polypeptide

The methods for decreasing the expression of LLG polypeptide to correctthose conditions in which LLG polypeptide activity contributes to adisease or disorder associated with an undesirable lipid profile includebut are not limited to administration of a composition comprising anantisense nucleic acid, administration of a composition comprising anintracellular binding protein such as an antibody, administration of acomposition comprising the LLGN polypeptide or another fragment of LLGand administration of a composition comprising a nucleic acid whichencodes the LLGN polypeptide or another fragment of LLG.

In one embodiment, a composition comprising an antisense nucleic acid isused to down-regulate or block the expression of LLG. In one preferredembodiment, the nucleic acid encodes antisense RNA molecules. In thisembodiment, the nucleic acid is operably linked to signals enablingexpression of the nucleic acid sequence and is introduced into a cellutilizing, preferably, recombinant vector constructs, which will expressthe antisense nucleic acid once the vector is introduced into the cell.Examples of suitable vectors includes plasmids, adenoviruses,adeno-associated viruses, retroviruses, and herpes viruses. Preferably,the vector is an adenovirus. Most preferably, the vector is areplication defective adenovirus comprising a deletion in the E1 and/orE3 regions of the virus.

In another embodiment, the expression of LLG is down-regulated orblocked by the expression of a nucleic acid sequence encoding anintracellular binding protein which is capable of selectivelyinteracting with LLG. WO 94/29446 and WO 94/02610, the contents of whichare incorporated herein by reference, disclose cellular transfectionwith genes encoding an intracellular binding protein. An intracellularbinding protein includes any protein capable of selectively interacting,or binding, with LLG in the cell in which it is expressed and ofneutralizing the function of bound LLG. Preferably, the intracellularbinding protein is an antibody or a fragment of an antibody. Morepreferably, the intracellular binding protein is a single chainantibody.

WO 94/02610 discloses preparation of antibodies and identification ofthe nucleic acid encoding a particular antibody. Using LLG or a fragmentthereof, a specific monoclonal antibody is prepared by techniques knownto those skilled in the art. A vector comprising the nucleic acidencoding an intracellular binding protein, or a portion thereof, andcapable of expression in a host cell is subsequently prepared for use inthe method of this invention.

Alternatively, LLG activity can be blocked by administration of aneutralizing antibody into the circulation. Such a neutralizing antibodycan be administered directly as a protein, or it can be expressed from avector (with a secretory signal).

In another embodiment, LLGXL activity is inhibited by the administrationof a composition comprising the LLGN polypeptide or another fragment ofLLG. This composition may be administered in a convenient manner, suchas by the oral, topical, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal, or intradermal routes. The composition may beadministered directly or it may be encapsulated (e.g. in a lipid system,in amino acid microspheres, or in globular dendrimers). The polypeptidemay, in some cases, be attached to another polymer such as serum albuminor polyvinyl pyrrolidone.

In another embodiment, LLGXL activity is inhibited through the use ofsmall molecular weight compounds, which interfere with its enzymaticproperties or prevent its appropriate recognition by cellular bindingsites.

In another embodiment, LLGXL activity is inhibited through the use ofgene therapy, that is, through the administration of a compositioncomprising a nucleic acid which encodes and directs the expression ofthe LLGN polypeptide or another fragment of LLG.

In a specific embodiment, the LLG gene of the present invention also hasan affinity for heparin. LLG polypeptide binding to extracellularheparin in the lumen of blood vessels would permit LLG to bind to andaccelerate LDL uptake by acting as a bridge between LDL and theextracellular heparin. In the localized area of an atheroscleroticlesion, an increased level of lipase activity is hypothesized toaccelerate the atherogenic process (Zilversmit, D. B. (1995) Clin. Chem.41, 153-158; Zambon, A., Torres, A., Bijvoet, S., Gagne, C., Moojani,S., Lupien, P. J., Hayden M. R., and Brunzell, J. D. (1993) Lancet 341,1119-1121). This may be due to an increase in the binding and uptake oflipoproteins by vascular tissue mediated by lipases (Eisenberg, S.,Sehayek, E., Olivecrona, T. Vlodaysky, I. (1992) J. Clin. Invest. 90,2013-2021; Tabas, I., Li, 1., Brocia R. W., Xu, S. W., Swenson T. L.Williams, K J. (1993) J. Biol. Chem. 268, 20419-20432; Nordestgaard, B.G., and Nielsen, A. G. (1994) Curr. Opin. Lipid. 5, 252-257; Williams, KJ., and Tabas, I. (1995) Art. Thromb. and Vasc. Biol. 15, 551-561).Additionally, a high local level of lipase activity may result incytotoxic levels of fatty acids and lysophosphatidylcholine beingproduced in precursors of atherosclerotic lesions. This particularactivity of LLG may contribute to the development or progression ofatherosclerosis, particularly in the context of excessive lipid levelsin a subject due to dietary or genetic factors. Thus, the presentinvention permits inhibition of lipoprotein accumulation by inhibitingLLG polypeptide expression or binding to lipoprotein (e.g., LDL).

2) Treatment of Undesirable Lipid Profiles Associated with InsufficientLLG

Polypeptide Activity

The methods for increasing the expression of LLG polypeptide to correctthose conditions in which LLG polypeptide activity contributes to adisease or disorder associated with an undesirable lipid profile includebut are not limited to administration of a composition comprising theLLGXL polypeptide and administration of a composition comprising anucleic acid which encodes the LLGXL polypeptide.

In one embodiment, the level of LLGXL activity is increased through theadministration of a composition comprising the LLGXL polypeptide. Thiscomposition may be administered in a convenient manner, such as by theoral, topical, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal, or intradermal routes. The composition may beadministered directly or it may be encapsulated (e.g. in a lipid system,in amino acid microspheres, or in globular dendrimers). The polypeptidemay, in some cases, be attached to another polymer such as serum albuminor polyvinyl pyrrolidone.

In another embodiment, the level of LLGXL is increased through the useof small molecular weight compounds, which can upregulate LLGXLexpression at the level of transcription, translation, orpost-translation.

In another embodiment, the level of LLGXL is increased through the useof gene therapy, that is, through the administration of compositioncomprising a nucleic acid which encodes and directs the expression ofthe LLGXL polypeptide.

Intrahepatic cholestasis can be characterized by increased serumcholesterol and phospholipid levels. A recently described, phalloidindrug-induced intrahepatic cholestasis model in rats demonstratedsignificant increases in the serum levels of cholesterol andphospholipid (Ishizaki, K, Kinbara, S., Miyazawa, N., Takeuchi, Y.,Hirabayashi, N., Kasai, H., and Araki, T. (1997) Toxicol. Letters 90,29-34). The products of this invention may be used to treat intrahepaticcholestasis in patients that have increased serum cholesterol and/orphospholipid. In addition, this rat model also exhibited a severedecrease in biliary cholesterol excretion rates. The LLG polypeptide andnucleic acid products of this invention may be used to treat patientswith an impaired biliary excretion system.

Intrahepatic cholestasis is also characterized by impaired bile flowfrom the liver. Recently, the loci for progressive familial intrahepaticcholestasis (PFIC or Byler disease) and benign recurrent intrahepaticcholestasis (BRJC) were mapped to 18q21-q22 (Carlton, V. E. H., Knisely,A. S., and Freimer, N. B. (1995) Hum. Mol. Genet. 4, 1049-1053 andHouwen, R. H., Baharloo, S., Blankenship, K., Raeymaekers, P., Juyn, J.,Sandkuijl, L. A., and Freimer, N. B. (1994) Nature Genet. 8, 380-386,respectively). As LLG gene maps within this chromosomal region at 18q21,the LLG gene or products of this invention may be used to treat patientswith intrahepatic cholestasis that is caused by a mutation or defectiveexpression of the PFIC/BRIC disease gene(s).

In another embodiment, the LLG gene or polypeptide products of thisinvention may be used to treat patients with intrahepatic cholestasisthat is not due to a defect in the PFIC/BRIC disease gene(s) at18q2′-q22. A recent study suggested that another locus, located outsideof the 18q21-q22 region may also produce the PFIC phenotype(Strautnieks, S. S., Kagalwalla, A. F., Tanner, M. S., Gardiner, R. M.,and Thompson, R. I. (1996) J. Med. Genet. 33, 833-836). Nevertheless,administration of LLG polypeptide, either directly or via gene therapy,may alleviate this form of the condition.

In gene therapy one or more nucleic acids encoding a polypeptide, aswell as regulatory regions controlling their expression, are transferredinto the target cells of a human or other animal. This transfer iscarried out either ex vivo in a procedure in which the nucleic acid istransferred to cells in the laboratory and the modified cells are thenadministered to the human or other animal, or in vivo in a procedure inwhich the nucleic acid is transferred directly to cells within the humanor other animal. The transfer of nucleic acids can be achieved usingeither the viral or non-viral vectors described above.

Non-viral vectors may be transferred into cells using any of the methodsknown in the art, including calcium phosphate coprecipitation,lipofection (synthetic anionic and cationic liposomes),receptor-mediated gene delivery, naked DNA injection, electroporationand bioballistic or particle acceleration.

EXAMPLES

The following examples illustrate the invention. These examples areillustrative only, and do not limit the scope of the invention.

Example 1 Identification of a Differentially Expressed cDNA

A) RNA Preparation

Human monocytic THP-1 cells (Smith, P. K., Krohn, R. I., Hermanson, G.T., Mallia, A. K., Gartner, F. H. Provenzano, M. D., Fujimoto, E. K.,Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150,76-85) were cultured in RPMI-1640 medium (GIBCO) with 25 mM HEPES, 10%fetal bovine serum, 100 units/ml penicillin G sodium and 100 units/mlstreptomycin sulfate. Cells were plated onto 15 cm tissue culture dishesat 1.5×10⁷ cells/plate, and treated with 40 ng/ml phorbol 12-myristate13-acetate (Sigma) for 48 hours to induce differentiation of the cells.Human low density lipoproteins (LDL) were purchased from Calbiochem, andwere dialyzed exhaustively versus PBS at 4° C. The LDL was then dilutedto 500 μg/ml and dialyzed versus 5 μM CuSO₄ in PBS at 37° C. for 16hours. To stop oxidation, the LDL was dialyzed exhaustively versus 150mM NaCl, 0.3 mM EDTA, then filter sterilized. Protein concentration wasdetermined by the BCA method (Schuh, J. Fairclough, G. F., andHaschemeyer, R. H. (1978) Proc. Natl. Acad. Sci. USA 75, 3173-3177)(Pierce). The degree of oxidation was determined by TBARS (Chomczynski,P. (1993) Biotechniques 15, 532-537), and was between 25-30 nmol MDAequivalents/mg protein. The differentiated THP-1 cells were exposed for24 hours to either 50 μg/ml oxidized LDL or NaCl-EDTA buffer in RPMImedium with 10% lipoprotein-deficient fetal bovine serum (Sigma). Toharvest the RNA, the plates were rinsed with 10 ml of PBS, then 14 ml ofTRIZOL (Liang, P. and Pardee, A. B. (1992) Science 257, 967-971) (GIBCO)were added to each plate. The solution was pipetted several times tomix, then like samples were pooled into centrifuge tubes and 3 mlchloroform per plate were added and mixed. The tubes were centrifugedfor 15 minutes at 12000×g. After centrifugation the upper layer wastransferred to a new tube and 7.5 ml isopropanol per plate was added andmixed. The tubes were centrifuged at 12000×g for 20 minutes. The pelletwas rinsed with ice-cold 70% ethanol and dried at room temperature. Thepellets were suspended in 500 μl TE (Tris-EDTA) and treated with 200units RNase-free DNAse 1 and 200 units RNasin placental RNase inhibitor(Promega) for 30 minutes at 37° C. The RNA was purified by sequentialextractions with phenol, phenol/chloroform/isoamyl alcohol (25:24:1),and chloroform/isoamyl alcohol (24:1) followed by ethanol precipitation.

B) cDNA Synthesis cDNA synthesis and PCR amplification were accomplishedusing protocols from the Differential Display Kit, version 1.0 (DisplaySystems Biotechnology, Inc.) This system is based on the techniqueoriginally described by Liang and Pardee (Mead, D. A., Pey, N. K.,Herrnstadt, C., Marcil, R. A., and Smith, L. M., (1991) Bio/Technology9, 657-663). The primer pairs which yielded the cDNA fragment containingthe first information of the lipase like gene were downstream primer 7and upstream primer 15. The cDNA for the amplification was synthesizedas follows, using RNA derived from PMA treated THP-1 cells exposed toeither buffer or oxidized LDL: 3 μl of 25 μM downstream primer 7 and 7.5μl of diethylpyrocarbonate (DEPC)-treated water were added to 300 ng(3.0 μl) RNA from either sample of THP-1 RNA. This was heated to 70° C.for 10 minutes then chilled on ice. To this tube were added 3 μl of5×PCR buffer (250 mM Tris-HCl pH 8.3, 375 mM KCl)(GIBCO), 3 μl 25 mMMgCl₂, 3 μl 0.1M DTT, 1.2 μl 500 μM dNTPs, 0.7 μl RNasin, and 5.6 μlDEPC-treated water. The tubes were incubated for 2 minutes at roomtemperature, after which 1.5 μl (300 units) Superscript II RNaseH-reverse transcriptase (GIBCO) were added. The tubes were incubatedsequentially at room temperature for 2 minutes, 60 minutes at 37° C.,and 5 minutes at 95° C., followed by chilling on ice. PCR amplificationwas performed using a master mix containing 117 μl 10×PCR buffer (500 mMKCl, 100 mM Tris-HCl pH 8.3, 15 mM MgCl₂, and 0.01% (w/v) gelatin), 70.2μl 25 mM MgCl₂, 5.9 μl alpha-³³P dATP (10 m Ci/ml, DuPont NEN), 4.7 μl500 μM dNTP mix, 11 μl AmpliTaq DNA polymerase (5 units/μl,Perkin-Elmer), and 493.3 μl DEPC-treated water. For each reaction, 12 μlof the master mix was added to 2 μl downstream primer #7, 1 μl of cDNA,and 5 μl of upstream primer #15. The reaction mixes were heated to 94°C. for 1 minute, then thermocycled 40 times with a denaturing step of94° C. for 15 seconds, annealing step of 40° C. for 1 minute, and anextension step of 72° C. for 30 seconds. Following the 40 cycles, thereactions were incubated at 72° C. for 5 minutes and stored at 10° C.The PCR reactions were performed in a Perkin-Elmer GeneAmp System 9600thermocycler.

Four microliters of the amplification reaction were mixed with an equalvolume of loading buffer (0.2% bromphenol blue, 0.2% Xylene cyanol, 10mM EDTA pH 8.0, and 20% glycerol). Four microliters of this mix was runon a 6% nondenaturing acrylamide sequencing format gel for 3 hours at1200 volts (constant voltage). The gel was dried at 80° C. for 1.5 hoursand exposed to Kodak XAR film. An amplification product found only inthe reaction containing cDNA from THP-1 cells exposed to oxidized LDLwas identified and excised from the gel. 100 μl of DEPC-treated waterwas added to a microcentrifuge tube containing the excised gel fragmentand was incubated for 30 minutes at room temperature followed by 15minutes at 95° C.

To reamplify the PCR product, 26.5 microliters of the eluted DNA wereused in a amplification reaction that also included 5 μl 10×PCR buffer,3 μl 25 mM MgCl₂, 5 μl 500 μM dNTPs, 5 μl 2 μM downstream primer 7, 7.5μl upstream primer 15, and 0.5 μl Amplitaq polymerase. The PCR cyclingparameters and instrument were as described above. Followingamplification, 20 μl of the reamplification was analyzed on an agarosegel and 4 μl was used to subclone the PCR products into the vector pCRIIusing the TA cloning system (Frohman, M. A., Dush, M. K., and Martin, G.R. (1988) Proc. Natl. Acad. Sci. USA 85, 8998-9002) (Invitrogen).Following an overnight ligation at 14° C., the ligation products wereused to transform E. coli. Resulting transformants were picked and 3 mlovernight cultures were used in plasmid minipreparations. Insert sizeswere determined using EcoRI digestions of the plasmids and clonescontaining inserts of the approximate size of the original PCR productwere sequenced using fluorecent dye-terminator reagents (Prism, AppliedBiosystems) and an Applied Biosystems 373 DNA sequencer. The sequence ofthe PCR product is shown in FIG. 2. The sequence of the amplificationprimers is underlined.

C) 5′ RACE Reaction

Extension of the cDNA identified through RT-PCR was accomplished usingthe 5′RACE system (Loh, E. Y., Eliot, J. F., Cwirla, S., Lanier, L. L.,and Davis, M. M. (1989) Science 243, 217-219; Simms, D., Guan, N., andSitaraman, K., (1991) Focus 13, 99) (GIBCO). One microgram of the THP-1RNA (oxidized LDL treated) used initially in the differential displayreactions was utilized in the 5′RACE procedure:

1 μl (1 μg) of RNA was combined with 3 μl (3 pmol) primer 2a and 11 μlDEPC-treated water and heated to 70° C. for 10 minutes followed by 1minute on ice. 2.5 μl 10× reaction buffer (200 mM Tris-HCl pH 8.4, 500mM KCl), 3 μl 25 mM MgCl₂, 1 μl 10 mM dNTP mix, and 2.5 μl 0.1 M DTTwere added. The mix was incubated at 42° C. for 2 minutes, then 1 μlSuperscript II reverse transcriptase was added. The reaction wasincubated for an additional 30 minutes at 42° C., 15 minutes at 70° C.,and on ice for 1 minute. One microliter of RNase H (2 units) was addedand the mixture was incubated at 55° C. for 10 minutes. The cDNA waspurified using the GlassMax columns (Sambrook, f. Fritsch, E. F., andManiatis, T. (1989) Molecular Cloning: A Laboratory Manual, secondedition, Cold Spring Harbor Laboratory Press, Plainview, N.Y.) includedin the kit. The cDNA was eluted from the column in 50 μl dH₂O,lyophilized, and resuspended in 21 μl dH₂O. Tailing of the cDNA wasaccomplished in the following reaction: 7.5 μl dH₂O, 2.5 μl reactionbuffer (200 mM Tris-HCl pH 8.4, 500 mM KCl), 1.5 μl 25 mM MgCl₂, 2.5 μl2 mM dCTP, and 10 μl of the cDNA were incubated at 94° C. for 3 minutes,then 1 minute on ice. 1 μl (10 units) of terminal deoxynucleotidyltransferase was added and the mixture was incubated for 10 minutes at37° C. The enzyme was heat inactivated by incubation at 70° C. for 10minutes and the mixture was placed on ice. PCR amplification of the cDNAwas performed in the following steps: 5 μl of the tailed cDNA wasincluded in a reaction which also contained 5 μl 10×PCR buffer (500 mMKCl, 100 mM Tris-HO pH 8.3, 15 mM MgCl₂, and 0.01% (w/v) gelatin), 1 μl10 mM dNTP mix, 2 μl (10 pmol) anchor primer, 1 μl (20 pmol) primer 3a,and 35 μl dH₂O. The reaction was heated to 95° C. for 1 minute, then 0.9μl (4.5 units) Amplitaq polymerase was added. The reaction was cycled 40times under the following conditions: 94° C. for 5 seconds, 50° C. for20 seconds, and 72° C. for 30 seconds. One microliter of this reactionwas used in a nested reamplification to increase levels of specificproduct for subsequent isolation. The reamplification included: 1 μlprimary amplification, 5 μl 10×PCR buffer, 1 μl 10 mM dNTP mix, 2 μl (20pmol) universal amplification primer, 2 μl (20 pmol) primer 4a, and 38μl dH₂O. The reaction was heated to 95° C. for 1 minute, then 0.7 μl(3.5 units) Amplitaq polymerase was added. The reaction was cycled 40times under these conditions; 94° C. for 5 seconds, 50° C. for 20seconds, and 72° C. for 30 seconds. The amplification products wereanalyzed via 0.8% agarose gel electrophoresis. A predominant product ofapproximately 1.2 kilobase pairs was detected. Two microliters of thereaction products were cloned into the pCRII vector from the TA cloningkit (Invitrogen) and incubated at 14° C. overnight. The ligationproducts were used to transform E. coli. The insert sizes of theresulting transformants were determined following EcoRI digestion.Clones containing inserts of the approximate size of the PCR productwere sequenced using fluorescent dye-terminator reagents (Prism, AppliedBiosystems) and an Applied Biosystems 373 DNA sequencer. The sequence ofthe RACE product including the EcoRI sites from the TA vector are shownin FIG. 3. The sequences of the amplimers (universal amplificationprimer and the complement to 5′RACE primer 4a) are underlined.

Example 2 Cloning and Chromosomal Localization of the LLGXL Gene

A) cDNA Library Screening

A human placental cDNA library (Oligo dT and random primed, Cat #5014b,Lot #52033) was obtained from Clontech (Palo Alto, Calif.). Aradiolabeled probe was created by excising the insert of a plasmidcontaining the 5′RACE reaction PCR product described above. The probewas radiolabeled using the random priming technique: the DNA fragment(50-100 ng) was incubated with 1 μg of random hexamers (Gibco) at 95° C.for 10 minutes followed by 1 minute on ice. At room temperature thefollowing were added: 3 μl 10× Klenow buffer (100 mM Tris-HCl pH 7.5, 50mM MgCL₂, 57 mM dithiothreitol; New England Biolabs), 3 μl 0.5 mM dATP,dGTP, dTTP), 100 μCi α-³²PdCTP (3000 Ci/mmol, New England Nuclear), and1 μl Klenow fragment of DNA polymerase I (5 units, Gibco). The reactionwas incubated for 2-3 hours at room temperature and the reaction wasthen stopped by increasing the volume to 100 μl with TE pH 8.0 andadding EDTA to a final concentration of 1 mM. The unincorporatednucleotides were removed by raising the reaction volume to 100 μl andpassing over a G-50 spin column (Boehringer Mannheim). The resultingprobes had a specific activity greater than 5×10⁸ cpm/μg DNA.

The library was probed using established methods (Walter, P., Gilmore,R., and Blobel, G. (1984) Cell 38, 5-8). Briefly, the filters werehybridized for 24 hours at 65° C. in 4.8×SSPE (20×SSPE=3.6 M NaCl, 0.2 MNaH₂PO₄, 0.02 M EDTA, pH 7.7), 20 ml Tris-HCl pH 7.6, 1×Denhardt'ssolution (100X=2% Ficoll 400, 2% polyvinylpyrrolidone, 2% BSA), 10%dextran sulfate, 0.1% SDS, 100 μg/ml salmon sperm DNA, and 1×10⁶ cpm/mlradiolabelled probe. Filters were then washed three times for 15 minutesat room temperature in 2×SSC (1×SSC=150 mM NaCl, 15 mM sodium citrate pH7.0), 0.1% sodium dodecyl sulfate (SDS) followed by three washes for 15minutes each at 65° C. in 0.5×SSC, 0.1% SDS. Phage which hybridized tothe probe were isolated and amplified. DNA was purified from theamplified phage using LambdaSorb reagent (Promega) according to themanufacturer's instructions. The inserts were excised from the phage DNAby digestion with EcoRI. The inserts were subcloned into the EcoRI siteof a plasmid vector (Bluescript II SK, Stratagene). The sequence of theopen reading frame contained within the 2.6 kb EcoRI fragment of thecDNA was determined by automated sequencing as described above. Thesequence is shown in FIG. 4. The amino acid sequence of the predictedprotein encoded by the open reading frame is shown in FIG. 5 and hasbeen termed LLGXL. The first methionine is predicted to be encoded bynucleotide pairs 252-254. The predicted protein is 500 amino acids inlength. The first 18 amino acids form a sequence characteristic of asecretory signal peptide (Higgins, D. G., and Sharp, P. M. (1988) Gene73, 237-244). The propeptide is predicted to have a molecular weight of56,800 Daltons. Assuming cleavage of the signal peptide at position 18,the unmodified mature protein has a molecular weight of 54,724 Daltons.

The overall similarities between this protein and the other knownmembers of the triacylglycerol lipase family is illustrated in FIG. 6Aand Table 1. In the alignment shown in FIGS. 6A-C, LLG is thepolypeptide (SEQ ID NO: 6) encoded by the cDNA (SEQ ID NO: 5) describedin Example 1, and hereafter referred to as LLGN. This protein isidentical with the LLGXL protein in the amino terminal 345 residues.Nine unique residues are followed by a termination codon, producing apropolypeptide of 39.3 kD and a mature protein of 37.3 kD. The sequenceswhich are common to LLGN and LLGXL are nucleic acid sequence SEQ ID NO:9 and amino acid sequence SEQ ID NO: 10.

Interestingly, the position at which the LLGN and LLGXL proteins divergeis at a region known from the structure of the other lipase to bebetween the amino and carboxy domains of the proteins. Therefore, theLLGN protein appears to consist of only one of the two domains oftriacylglycerol lipases. This sequence contains the characteristic“GXSXG” lipase motif at positions 167-171 as well as conservation of thecatalytic triad residues at Sex 169, Asp 193, and H is 274. Conservationof cysteine residues (positions 64, 77, 252, 272, 297, 308, 311, 316,463, and 483) which have been implicated in disulfide linkage in theother lipases suggests that the LLGXL protein has structuralsimilarities to the other enzymes. There are five predicted sites forN-linked glycosylation; at amino acid positions 80, 136, 393, 469, and491. The protein sequences used in the comparisons are human lipoproteinlipase (LPL; Genbank accession #M15856, SEQ ID NO: 13), Human hepaticlipase (HL; Genbank accession #J03540, SEQ ID NO: 14), human pancreaticlipase (PL; Genbank accession # M93285, SEQ ID NO: 15), human pancreaticlipase related protein-1 (PLRP-1; Genbank accession # M93283), and humanpancreatic lipase related protein-2 (PLRP-2; Genbank accession #M93284).

TABLE 1 Similarity of triacylglycerol lipase gene family LLGXL LPL HL PLPLRP1 PLRP2 LLGXL — 42.7 36.5 24.5 22.5 22.6 LPL 42.7 — 40.0 22.8 22.720.9 HL 36.5 40.0 — 22.8 24.0 22.0 PL 24.5 22.8 22.8 — 65.2 62.2 PLRP122.5 22.7 24.0 65.2 — 61.7 PRLP2 22.6 20.9 22.0 62.2 61.7 —Percent similarity was based on pairwise alignment using the Clustalalgorithm (Camps, L., Reina, M., Llobera, M., Vilaro, S., andOlivecrona, T. (1990) Am. J. Physiol. 258, C673-C681) in the Megalignprogram of the Lasergene Biocomputing Software Suite (Dnastar).B) Chromosomal Localization

DNA from a P1 clone (Sternberg, N., Ruether, J. and DeRiel, K. The NewBiologist 2:151-62, 1990) containing genomic LLG DNA was labelled withdigoxigenin UTP by nick translation. Labelled probe was combined withsheared human DNA and hybridized to PHA stimulated peripheral bloodlymphocytes from a male donor in a solution containing 50% formamide,10% dextran sulfate, and 2×SSC. Specific hybridization signals weredetected by incubating the hybridized cells in fluoresceinatedantidigoxigenin antibodies followed by counterstaining with DAPI. Thisinitial experiment resulted in specific labeling of a group Echromosome, which was believed to be chromosome 18 on the basis of DAPIstaining.

A second experiment was conducted in which a biotin labelled probespecific for the centromere of chromosome 18 was cohybridized with theLLG probe. This experiment resulted in the specific labeling of thechromosome 18 centromere in red and the long arm of chromosome 18 ingreen. Measurements of 11 specifically labelled hybridized chromosomes18 demonstrated that LLG has a Fiter of 0.67 (Franke measurement of0.38), which corresponds to band 18q21. Several genetic diseases,including intrahepatic cholestasis, cone rod dystrophy, and familialexpansile osteolysis, are believed to involve defects in thischromosomal region.

Example 3 LLG RNA Analysis

A) Expression of LLG RNA in THP-1 Cells

Analysis of the mRNA from which the cDNA was derived was performed bynorthern analysis of THP-1 RNA. RNA from these cells was prepared asdescribed above. The mRNA was purified from the total RNA through theuse of a poly-dT-magnetic bead system (Polyattract system, Promega).Three micrograms of poly (A)-containing mRNA was electrophoresed on a 1%agarose-formaldehyde gel. The gel was washed for 30 minutes in dH₂O.RNAs were vacuum transferred to a nylon membrane using alkaline transferbuffer (3M NaCl, 8 mM NaOH, 2 mM sarkosyl). After transfer, the blot wasneutralized by incubation for 5 minutes in 200 mM phosphate buffer pH6.8. The RNA was crosslinked to the membrane using an ultravioletcrosslinker apparatus (Stratagene).

A probe was made by excising the insert of a plasmid containing the5′RACE reaction PCR product described above. The probe was radiolabeledusing the random priming technique described in Example 2.

The filters were prehybridized in QuikHyb rapid hybridization solution(Stratagene) for 30 minutes at 65° C. The radiolabeled probe (1-2×10⁶cpm/ml) and sonicated salmon sperm DNA (final concentration 100 μg/ml)were denatured by heating to 95° C. for 10 minutes and quick-chilled onice before adding to the filter in QuikHyb. Hybridization was for 3hours at 65° C. The unhybridized probe was removed by washing thefilters two times for 15 minutes with 2×SSC, 0.1% sodium dodecyl sulfateat room temperature followed by two times for 15 minutes in 0.1×SSC,0.1% SDS at 62° C. Following the washes, the filters were allowed to drybriefly and then exposed to Kodak XAR-2 film with intensifying screensat −80° C. The results are shown in FIG. 7, which shows a major mRNAspecies of approximately 4.5 kilobases. Minor species of 4.3 and 1.6kilobases are also present. The expected size of the LLGN cDNA is 1.6kb. The LLGXL sequence is likely to be encoded by the major species ofmRNA detected.

B) Expression of LLG RNA in Various Human Tissues.

A commercially prepared filter containing 3 μg each of mRNAs from humantissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney,and pancreas) was obtained from Clontech (Catalog #7760-1). This filterwas probed and processed as described above. After probing with theradiolabeled LLG fragment and autoradiography, the probe was stripped bywashing in boiling 0.1×SSC, 0.1% SDS for 2×15 min. in a 65° C.incubator. The membranes were then probed with a 1.4 kilobase pair DNAfragment encoding human lipoprotein lipase. This fragment was obtainedby RT-PCR of the THP-1 RNA (PMA and oxLDL treated) using the 5′LPL and3′LPL primers described in FIG. 1 and the RT-PCR conditions describedabove. After autoradiography, the membranes were stripped again andreprobed with a radiolabeled fragment of the human beta actin cDNA tonormalize for RNA content. The results of these analyses are shown inFIG. 8. The highest levels of LLG message were detected in placentalRNA, with lower levels found in RNAs derived from lung, liver, andkidney tissue. In agreement with previous studies by others (Verhoeven,A. J. M., Jansen, H. (1994) Biochem. Biophys. Acta 1211, 121-124),lipoprotein lipase message was found in many tissues, with highestlevels found in heart and skeletal muscle tissue. Results of thisanalysis indicates that the tissue distribution of LLG expression isvery different from that of LPL. The pattern of LLG expression is alsodifferent from that of either hepatic lipase or pancreatic lipase, asreported by others (Wang, C.-S., and Hartsuck, J. A. (1993) Biochem.Biophys. Acta 1166, 1-19; Semenkovich, C. F., Chen, S.-W., Wims, M., LuoC.-C., L₁, W.-H., and Chan, L. (1989) J. Lipid Res. 30, 423-431; Adams,M. D., Kerlavage, A. R., Fields, C., and Venter, C. (1993) Nature Genet.4, 256-265).

To determine the expression pattern in additional human tissues, anothercommercially prepared membrane was probed with LLGXL cDNA. This dot blot(Human RNA Master Blot, Clontech Cat. #7770-1) contains 100-500 ng mRNAfrom 50 different tissues and is normalized for equivalent housekeepinggene expression (Chen, L., and Morin, R. (1971) Biochim. Biophys. Acta231, 194-197). A 1.6 kb DraI-SrfI fragment of the LLGXL cDNA was labeledwith ³²PdCTP using a random oligonucleotide priming system (Prime It II,Stratagene) according to the manufacturer's instructions. After 30minutes prehybridization at 65° C., the probe was added to QuikHybhybridization solution at 1.3×10⁶ cpm/ml. Hybridization was for 2 hoursat 65° C. The unhybridized probe was removed by washing the filters twotimes for 15 minutes with 2×SSC, 0.1% sodium dodecyl sulfate at roomtemperature followed by two times for 15 minutes in 0.1×SSC, 0.1% SDS at62° C. Following the washes, the filters were allowed to dry briefly andthen exposed to Kodak XAR-2 film with intensifying screens at −80° C.for varying amounts of time. The resulting images were quantitated bydensitometry. The results are shown in Table 2. The relative expressionlevels of tissues represented in boththe multiple tissue northern andthe multiple tissue dot blot are similar, with highest levels inplacenta, and lower levels in lung, liver and kidney. Fetal liver,kidney, and lung also express roughly the same levels as the adulttissues. Surprisingly, thyroid tissue expression levels were the highestof all tissues represented, with expression of 122% of that in placentaltissue. While there is precedence for lipase expression by the placenta(Rothwell, J. E., Elphick, M. C. (1982) J. Dev. Physiol. 4, 153-159;Verhoeven, A. J. M., Carling D., and Jansen H. (1994) J. Lipid Res. 35,966-975; Burton, B. K., Mueller, H. W. (1980) Biochim. Biophys. Acta618, 449-460), the thyroid was not previously known to express anylipase. These results suggest that LLG expression may be involved inmaintenance of the placenta, where LLG may serve to liberate free fattyacids from substrates such as phospholipids as a source of energy. TheLLG expressed in the thyroid may provide precursors for the synthesis ofbioactive molecules by that gland.

TABLE 2 Expression of LLG mRNA in various human tissues whole brain N.D.substantial N.D. uterus N.D. mammary N.D. lung 29 nigra gland amygdalaN.D. temporal N.D. prostate 5 kidney 44 trachea 12 lobe caudate N.D.thalamus N.D. stomach N.D. liver 61 placenta 100 nucleus cerebellum 4sub-thalamic N.D. testes 9 small  6 fetal brain 5 nucleus intestinecerebral N.D. spinal cord N.D. ovary N.D. spleen N.D. fetal heart N.D.cortex frontal lobe N.D. heart N.D. pancreas N.D. thymus N.D. fetalkidney 56 hippocampus N.D. aorta N.D. pituitary N.D. peripheral N.D.fetal liver 14 gland leukocyte medulla N.D. skeletal N.D. adrenal N.D.lymph node N.D. fetal spleen N.D. oblongata muscle gland occipital N.D.colon 8 thyroid 122  bone marrow N.D. fetal thymus N.D. lobe glandputamen N.D. bladder N.D. salivary N.D. appendix  7 fetal lung 8 glandValues given are percentage of expression with levels in placentaltissue arbitrarily set at 100%. Values are average of densitometricmeasurements from two autoradiographic exposures. N.D. = not detectable.C) Expression of LLG RNA in Cultured Endothelial Cells.

Human umbilical vein endothelial cells (HUVEC) and human coronaryarterial endothelial cells (HCAEC) were obtained from Clonetics. HUVECswere propagated in a commercially prepared endothelial cell growthmedium (EGM, Clonetics) supplemented with 3 mg/ml bovine brain extract(Maciag, T., Cerundolo, J., Dsley, S., Kelley, P. R., and Forand, R.(1979) Proc. Natl. Acad. Sci. USA 76, 5674-5678), Clonetics), whileFICAECs were propagated in EGM supplemented with 3 mg/ml bovine brainextract and 3% fetal bovine serum (5% final concentration). Cells weregrown to confluence, then the medium was changed to EGM without bovinebrain extract. Cultures were stimulated by adding 100 ng/ml of phorbolmyristate (Sigma). After 24 hours incubation, the RNAs were extractedfrom the cells via the Trizol method described above. Twenty microgramsof total RNA was electrophoresed and transferred to the membrane foranalysis. The membranes were probed with LLG and LPL probes as describedabove. The results are shown in FIG. 9. Twenty micrograms of total RNAfrom THP-1 cells stimulated with PMA was run on the blot for comparison.RNA hybridizing to the LLG probe was detected in unstimulated and PMAstimulated HUVEC cells. In contrast, detectable levels of LLG mRNA wereonly found in HCAEC cultures after stimulation with PMA. In agreementwith previous studies of others, no detectable lipoprotein lipase mRNAwas detected in any of the endothelial RNAs (Verhoeven, A. J. M.,Jansen, H. (1994) Biochem. Biophys. Acta 1211, 121-124).

Example 4 LLG Protein Analysis

A) Antibody Preparation.

Antisera were generated to peptides with sequences corresponding to aregion of the predicted protein encoded by the LLG cDNA open readingframe. This peptide was chosen because of its high predictedantigenicity index (Jameson B. A., and Wolf, H. (1988) Comput. Applic.in the Biosciences 4, 181-186). The sequence of the immunizing peptidewas not found in any protein or translated DNA sequence in the Genbankdatabase. Its corresponding position in the LLG protein is shown in FIG.10. The carboxy terminal cysteine of the peptide does not correspond tothe residue in the LLG putative protein, but was introduced tofacilitate coupling to the carrier protein. The peptide was synthesizedon a Applied Biosystems Model 433A peptide synthesizer. Two milligramsof peptide was coupled to two milligrams of maleimide-activated keyholelimpet hemocyanin following the protocols included in the ImjectActivated Immunogen Conjugation Kit (Pierce Chemical). After desalting,one-half of the conjugate was emulsified with an equal volume ofFreund's complete adjuvant (Pierce). This emulsification was injectedinto a New Zealand White rabbit. Four weeks after the initialinoculation, a booster inoculation was made with an emulsification madeexactly as described above except Freund's incomplete adjuvant (Pierce)was used. Two weeks after the boost, a test bleed was made and titers ofspecific antibodies were determined via ELISA using immobilized peptide.A subsequent boost was made one month after the first boost.

B) Western Analysis of Medium from Endothelial Cell Cultures

HUVEC and HCEAC cells were cultured and stimulated with PMA as describedin Example 3C, except that the cells were stimulated with PMA for 48hours. Samples of conditioned medium (9 ml) were incubated with 500 μlof a 50% slurry of heparin-Sepharose CL-6B in phosphate buffered saline(PBS, 150 mM sodium chloride, 100 mM sodium phosphate, pH 7.2).Heparin-Sepharose was chosen to partially purify and concentrate the LLGproteins because of the conservation of residues in the LLGXL sequencewhich have been identified as critical for the heparin-binding activityof LPL (Ma, Y., Henderson, H. E., Liu, M.-S., Zhang, H., Forsythe, I. J.Clarke-Lewis, I., Hayden, M. R., and Brunzell, J. D. J. Lipid Res. 35,2049-2059; and FIGS. 6A-C). After rotation at 4° C. for 1 hour, thesamples were centrifuged for 5 minutes at 150×g. The medium wasaspirated and the Sepharose was washed with 14 ml PBS. Aftercentrifugation and aspiration, the pelleted heparin-Sepharose wassuspended in 200 μ12×SDS loading buffer (4% SDS, 20% glycerol. 2%β-mercaptoethanol, 0.002% bromophenol blue, and 120 mM Tris pH 6.8). Thesamples were heated to 95° C. for 5 minutes and 40 μl was loaded onto a10% Tris-Glycine SDS gel. After electrophoresis at 140 V forapproximately 90 minutes, the proteins were transferred tonitrocellulose membranes via a Novex electroblotting apparatus (210 V, 1hour). The membranes were blocked for 30 minutes in blocking buffer (5%nonfat dried milk, 0.1% Tween 20, 150 mM sodium chloride, 25 mM Tris pH7.2). Antipeptide antisera and normal rabbit serum was diluted 1:5000 inblocking buffer and was incubated with the membranes overnight at 4° C.with gentle agitation. The membranes were then washed 4×15 minutes withTBST (0.1% Tween 20, 150 mM sodium chloride, 25 mM Tris pH 7.2). Goatanti-rabbit peroxidase conjugated antisera (Boehringer Mannheim) wasdiluted 1:5000 in blocking buffer and incubated with the membrane for 1hour with agitation. The membranes were washed as above, reacted withRenaissance chemiluminescent reagent (DuPont NEN), and exposed to KodakXAR-2 film. The results are shown in FIG. 11. Two species ofimmunoreactive proteins are present in the samples from unstimulatedHUVEC and HCAEC cells. Levels of immunoreactive protein in theunstimulated HCAEC samples are much lower than the corresponding HUVECsample. Upon stimulation with PMA, three immunoreactive proteins aresecreted by the endothelial cell cultures. PMA exposure greatlyincreased the level of LLG proteins produced by the HCAEC cultures. PMAinduction of LLG proteins was not as dramatic in the HUVEC cultures.

Example 5 Recombinant LLG Protein Production

A) LLG Expression Constructs

The cDNAs encoding the LLGN and LLGXL proteins were cloned into themammalian expression vector pcDNA3 (Invitrogen). This vector allowsexpression of foreign genes in many mammalian cells through the use ofthe cytomegalovirus major late promoter. The LLGN 5′RACE product wascloned into the EcoRI site of pcDNA3. The LLGXL cDNA was digested withDraI and SrfI to yield a 1.55 kb cDNA (see FIG. 4.). The vector wasdigested with the restriction enzyme EcoRV and the vector and insertwere ligated using T4 DNA ligase and reagents from the Rapid LigationKit (Boehringer Mannheim) according to the manufacturers instructions.The ligation products were used to transform competent E. coli.Resultant colonies were screened by restriction analysis and sequencingfor the presence and orientation of the insert in the expression vector.

B) Transient Transfection of LLG in COS-7 Cells.

The LLG expression vectors were introduced into COS-7 cells through theuse of Lipofectamine cationic lipid reagent (GIBCO). Twenty-four hoursbefore the transfection, COS-7 cells were plated onto 60 mm tissueculture dishes at a density of 2×10⁵ cells/plate. The cells werepropagated in Dulbccco's modified Eagle's medium (DMEM; GIBCO)supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 μg/mlstreptomycin. One microgram of plasmid DNA was added to 300 μl ofOptimem I serum-free medium (Gibco). Ten microliters of Lipofectaminereagent were diluted into 300 μl of Optimem I medium and this wascombined with the DNA solution and allowed to sit at room temperaturefor 30 minutes. The medium was removed from the plates and the cellswere rinsed with 2 ml of Optimem medium. The DNA-Lipofectamine solutionwas added to the plates along with 2.7 ml Optimem medium and the plateswere incubated for 5 hours at 37° C. After the incubation, the serumfree medium was removed and replaced with DMEM supplemented with 2% FBSand antibiotics. Twelve hours post-transfection, some of the cultureswere treated with either 0.25 mM Pefabloc S. C. (Boehringer Mannheim), aprotease inhibitor, or 10 U/ml heparin. Thirty minutes before harvest,the heparin treated samples were treated with an additional 40 Untilheparin. The medium was removed from the cells 60 hours aftertransfection. Heparin-Sepharose CL-4B (200 μl of a 50% slurry in PBS pH7.2) was added to 1 ml of medium and was mixed at 4° C. for 1 hour. TheSepharose was pelleted by low speed centrifugation and was washed threetimes with 1 ml cold PBS. The Sepharose was pelleted and suspended in100 μl 2× loading buffer. The samples were heated to 95° C. for 5minutes. 40 μl of each sample was loaded onto a 10% SDS-PAGE gel.Electrophoresis and western analysis was performed using the anti-LLGantiserum as described above. The results are shown in FIG. 12. Proteinsfrom HCAEC conditioned medium were included for size references. LLGNmigrates at approximately 40 kD, corresponding to the lowest band inHCAEC. The medium from COS cells transfected with LLGXL cDNA containsboth 68 kD and 40 kD species. When these cells were treated withheparin, the amount of both 68 kD and 40 kD proteins recovered from themedium increased dramatically, indicating either the release ofproteoglycan-bound protein from the cell surface or stabilization of theproteins by heparin. When the cells were treated with the proteaseinhibitor Pefabloc, the amount of 68 kD protein increased relative tothat of the 40 kD species. This suggests that the lower molecular weightprotein produced by these cells is a proteolysis product of the larger68 kD form. The role of the mRNA identified through differential displaywhich encodes a shorter, 40 kD species is not known. There has, however,been a report of an alternately-spliced form of hepatic lipase whichapparently is expressed in a tissue-specific manner and would create atruncated protein.

Example 6 LLG in Animal Species

A) Cloning the Rabbit Homolog of LLG

A commercially available lambda cDNA library derived from rabbit lungtissue (Clontech. Cat. #TL1010b) was used to isolate a fragment of therabbit homolog of the LLG gene. Five microliters of the stock librarywere added to 45 μl water and heated to 95° C. for 10 minutes. Thefollowing were added in a final volume of 100 μl: 200 μM dNTPs, 20 mMTris-HCl pH 8.4, 50 mM KCl, 1.5 mM MgCl₂, 100 μM each primer DLIP774 andLLGgen2a, and 2.5 U Taq polymerase (GIBCO). The reaction wasthermocycled 35 times with the parameters of: 15 seconds at 94° C., 20seconds at 50° C. and 30 seconds at 72° C. Ten microliters of thereaction was analyzed via agarose gel electrophoresis. A product ofapproximately 300 basepairs was detected. A portion (4 μl) of thereaction mix was used to clone the product via the TA cloning system.The insert of a resulting clone was sequenced (SEQ ID NO: 11). Analignment between the deduced rabbit amino acid sequence (SEQ ID NO: 12)and the corresponding sequence of the human cDNA is also shown in FIG.14. Of the nucleotides not part of either amplification primer, there isan 85.8% identity between the rabbit and human LLG sequences. Thepredicted protein encoded by this rabbit cDNA shares 94.6% identity withthat of the human protein, with most of the nucleotide substitutions inthe third or “wobble” positions of the codons. Notably, this regionspans the “lid” sequence of the predicted LLG proteins and is a variabledomain in the lipase gene family. This is evidence that there is a highdegree of conservation of this gene between species.

B) LLG in Other Species

To demonstrate the presence of LLG genes in other species, genomic DNAsfrom various species were restriction digested with EcoRI, separated byelectrophoresis in agarose gels, and blotted onto nitrocellulosemembranes.

The membranes were hybridized overnight at 65° C. with 2.5×10⁶ cpm/ml ofrandom primed ³²P-LLG or ³²P-LPL (lipoprotein lipase) probe in ahybridization solution of 6×SSC, 10% dextran sulfate, 5×Dendardt'ssolution, 1% SDS, and 5 μg/ml salmon sperm DNA. The membranes werewashed with 0.1×SSC, 0.5% SDS for ten minutes at room temperature, thensequentially for ten minutes at 40° C., 50° C., and 55° C.Autoradiograms of the blots are shown in FIG. 16.

FIG. 16 shows the presence of LLG and LPL genes in all species examined,with the exception that no hybridization was observed with the LLG probeagainst rat DNA. The exceptional data from rat may represent an artifactcaused by generation of abnormally sized restriction fragmentscontaining LLG sequences. Such fragments may be outside of thefractionation range of the agarose gel or may blot inefficiently. Thedifferent bands detected by the two probes indicate that LPL and LLG areseparate, evolutionarily conserved genes.

Example 7 Enzymatic Activity of Llgxl

A) Phospholipase Activity

Conditioned media from COS-7 cells transiently expressing humanlipoprotein lipase (LPL), LLGN, or LLGXL were assayed for phospholipaseactivity. MEM containing 10% FBS (MEM) was used as the blank, andconditioned media from COS-7 cells transfected with an antisense LLGXLplasmid (AS) was used as a negative control.

A phosphatidylcholine (PC) emulsion was made up using 10 μlphosphatidylcholine (10 mM), 40 μl ¹⁴C-phosphatidylcholine, dipalmitoyl(2 μCi), labelled at the sn 1 and 2 positions, and 100 μl Tris-TCNB [100mM Tris, 1% Triton, 5 mM CaCl₂, 200 mM NaCl, 0.1% BSA). The emulsion wasevaporated for 10 minutes, then brought to a final volume of 1 ml inTris-TCNB.

Reactions were performed in duplicate and contained 50 μl PC emulsionand 950 μl medium. Samples were incubated in a shaking water bath for2-4 hours at 37° C. The reactions were terminated by adding 1 ml 1N HCl,then extracted with 4 ml of 2-propanol:hexane (1:1). The upper 1.8 mlhexane layer was passed through a silica gel column, and the liberated¹⁴C-free fatty acids contained in the flow-thru fraction werequantitated in a scintillation counter. The results of these assays areshown in FIG. 14.

B) Triacylglycerol Lipase Activity

Conditioned media from COS-7 cells transiently expressing humanlipoprotein lipase (LPL), LLGN, or LLGXL were assayed for triglycerollipase activity. MEM containing 10% FBS was used as the blank, andconditioned media from COS-7 cells transfected with an antisense LLGXLplasmid (AS) was used as a negative control.

A concentrated substrate was prepared as an anhydrous emulsion oflabeled triolein, [9,10-³H(N)] and unlabeled triolein (final totaltriolein=150 mg with 6.25×10⁸ cpm), which was stabilized by adding 9 mgof lecithin in 100% glycerol. 0.56 ml of ³H-triolein, (0.28 mCi) wasmixed with 0.17 ml of unlableled triolein and 90 μl of lecithin (9 mg).The mixture was evaporated under a stream of nitrogen. The dried lipidmixture was emulsified in 2.5 ml 100% glycerol by sonication (30 secondpulse level 2 followed by 2 second chill cycles over 5 minutes).

The assay substrate was prepared by dilution of 1 volume of concentratedsubstrate with 4 volumes of 0.2M Tris-HCl buffer (pH 8.0) containing 3%w/v fatty acid free bovine serum albumin. The diluted substrate wasvortexed vigorously for 5 seconds.

Reactions were performed in duplicate in a total volume of 0.2 mlcontaining 0.1 ml of assay substrate and 0.1 ml of the indicatedconditioned media. The reactions were Incubated for 90 minutes at 37° C.The reactions were terminated by adding 3.25 ml ofmethanol-chloroform-heptane 1.41:1.25:1 (v/v/v) followed by 1.05 ml of0.1M potassium carbonate-borate buffer (pH 10.5). After vigorous mixingfor 15 seconds, the samples were centrifuged for 5 minutes at 1000 rpm.A 1.0 ml aliquot of the upper aqueous phase was counted in ascintillation counter. The results of these assays are shown in FIG. 15.

Example 8 Use of LLG Polypeptide to Screen for Agonists or Antagonists

Recombinant LLG is produced in baculovirus-infected insect cells.Recombinant LLG is purified from the serum-containing or serum-freeconditioned medium by chromatography on heparin-Sepharose, followed bychromatography on a cation exchange resin. A third chromatographic step,such as molecular sieving, is used in the purification of LLG if needed.During purification, anti-peptide antibodies are used to monitor LLGprotein and the phospholipase assay is used to follow LLG activity.

In the fluorescent assay, the final assay conditions are approximately10 mM Tris-HCl (pH 7.4), 100 mM KCl, 2 mM CaCl, 5 μMC₆NBD-PC{1-acyl-2-[6-(nitro-2,1,3-benzoxadiazol-4-yl)amino]caproylphosphatidylcholine,and LLG protein (approx 1-100 ng). The reaction is subjected tofluorescence excitation at 470 nm, and enzyme activity, as measured bythe fluorescence emission at 540 nm is continuously monitored. Compoundsand/or substances to be tested for stimulation and/or inhibition of LLGactivity are added as 10-200 mM solutions in dimethylsulfoxide.Compounds which stimulate or inhibit LLG activity are identified ascausing an increased or decreased fluorescence emission at 540 nm.

In the thio assay, the final assay conditions are approximately 25 mMTris-HCl (pH 8.5), 100 mM KCl, 10 mM CaCl₂, 4.24 mM Triton X-100, 0.5 mM1,2-bis(hexanoylthio)-1,2-dideoxy-sn-glycero-3-phosphorylcholine, 5 mM4,4′-dithiobispyridine (from a 50 mM stock solution in ethanol), and1-100 ng recombinant LLG. Phospholipase activity is determined bymeasuring the increase in absorption at 342 μm. Compounds and/orsubstances to be tested for stimulation and/or inhibition of LLGactivity are added as 10-200 mM solutions in dimethylsulfoxide.Compounds which stimulate or inhibit LLG activity are identified ascausing an increased or decreased absorption at 342 nm.

Example 9 Transgenic Mice Expressing Human LLG

To further study the physiological role of LLG, transgenic miceexpressing human LLG are generated.

The 1.53 kb DraI/SrfI restriction fragment encoding LLGXL (see FIG. 4)was cloned into a plasmid vector (pHMG) downstream of the promoter forthe ubiquitously expressed 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase gene. Transgenic mice expressing different levels ofhuman LLGXL are generated using standard methods (see, e.g., G. L. Trompet al. Gene 1565:199-205, 1995). The transgenic mice are used todetermine the impact of LLGXL overexpression on lipid profile, vascularpathology, rate of development and severity of atherosclerosis, andother physiological parameters.

Example 10 Expression of LLG in Atherosclerotic Tissues

LLGXL expression in atherosclerosis was examined by performing a reversetranscription-polymerase chain reaction (RT-PCR) using mRNA isolatedfrom vascular biopsies from four patients with atherosclerosis. Thetissue samples were from the aortic wall (one sample), the iliac artery(two samples), and the carotid artery (one sample).

Atherosclerosis biopsies were received from Gloucestershire RoyalHospital, England, and polyA+mRNA was prepared and resuspended indiethylpyrocarbonate (DEPC) treated water at a concentration of 0.5μg/μl mRNA. Reverse transcriptase reactions were performed according tothe GibcoBRL protocol for Superscript Preamplification System for FirstStrand cDNA Synthesis. Briefly, the cDNA was synthesized as follows: 2μl of each mRNA was added to 1 μl oligo (dT)₁₂₋₁₈ primer and 9 μl ofDEPC water. The tubes were incubated at 70° C. for 10 minutes and put onice for 1 minute. To each tube, the following components were added: 2μl 10×PCR buffer, 2 μl 25 mM MgCl₂, 1 μl 10 mM dNTP mix and 2 μl 0.1MDTT. After 5 minutes at 42° C. 1 μl (200 units) of Super Script IIreverse transcriptase was added. The reactions were mixed gently, thenincubated at 42° C. for 50 minutes. The reactions were terminated byincubation at 70° C. for 15 minutes then put on ice. The remaining mRNAwas destroyed by the addition of 1 μl of RNase H to each tube andincubated for 20 minutes at 37° C.

PCR amplifications were performed using 2 μl of the cDNA reactions. Toeach tube the following were added: 5 μl 10×PCR buffer, 5 μl 2 mM dNTPs,1 μl hllg-gsp1 primer (20 pmol/ml. see FIG. 1), 1 μl hllg-gsp2a primer(20 pmol/ml, see FIG. 1), 1.5 μl 50 mM MgCl₂ 0.5 μl Taq polymerase (5U/ml) and 34 μl water. After holding the reactions at 95° C. for 2minutes, thirty cycles of PCR were performed as follows: 15 seconds at94° C., 20 seconds at 52° C., and 30 seconds at 72° C. The finishedreactions were held for 10 minutes at 72° C. before analysis by agarosegel electrophoresis. The hllg-gsp primers are specific for LLG and yieldan expected product of 300 bp. In a parallel PCR to show that the cDNAsynthesis reactions had been successful, primers specific for thehousekeeping gene, G3PDH (human glyceraldehyde 3-phosphatedehydrogenase) were used (1 μl each at 20 pmol/ml).

The G3PDH primers (see FIG. 1) yielded the expected product of 983 bp inall four vascular biopsy samples. LLG expression was detected in threeof the four samples, with no expression being detected in the carotidartery sample.

All the references discussed herein are incorporated by reference.

One skilled in the art will readily appreciate the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The peptides,polynucleotides, methods, procedures and techniques described herein arepresented as representative of the preferred embodiments, and intendedto be exemplary and not intended as limitations on the scope of thepresent invention. Changes therein and other uses will occur to those ofskill in the art which are encompassed within the spirit of theinvention or defined by the scope of the appended claims.

1. A method of improving the serum lipid profile of a human or otheranimal having an undesirable lipid profile comprising administration ofan effective amount of a composition comprising: (A) an antibody capableof specifically binding to an isolated polypeptide encoded by a lipaselike gene, wherein the polypeptide: (i) binds heparin, (ii) has homologywith human lipoprotein lipase and hepatic lipase, (iii) comprises a 39kD catalytic domain of the triacylglycerol lipase family, and (iv)comprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10; and (B) apharmaceutically acceptable carrier.
 2. The method of claim 1 whereinthe antibody is able to neutralize a phospholipase activity of thepolypeptide.
 3. The method of claim 1 wherein the antibody is amonoclonal antibody.
 4. The method of claim 1 wherein the antibody is apolyclonal antibody.